pediatric diagnosis of hiv-1 infection using dried blood spots

Pediatric Diagnosis of HIV-1 Infection Using Dried Blood SpotsChin-Yih Ou, PhDNCHSTP/DHAPCenters for Disease Control and Prevention

ObjectivesTo develop and validate a nucleic acid based-assay for the diagnosis of HIV-1 infection in infants and young children in resource-poor countries using dried blood spots

Mother-to-Child Transmission: Crisis

Without antiretroviral intervention, 20-45% of HIV-infected women transmit HIV to infants.

In 2004, between 650,000 to 750,000 children were newly infected. About half a million of children died of AIDS.

Because antiretroviral drugs are becoming more affordable, many developing countries are expanding programs on prevention of mother-to-child transmission. It is thus important to identify infected infants early to initiate antiretroviral therapy.

Problems of Laboratory Tests for the diagnosis of HIV-1 vertical transmission

Because of the presence of maternal antibodies in children under the age of 18 months, serologic tests are not useful.

Enhanced HIV p24 assay is potentially useful, but remains to be validated.

Nucleic Acid Technology (NAT) based Assays could be useful in pediatric diagnosis:

Standard PCR testing on whole blood, cell pellets, and dried blood spots (DBS) has been used; but each approach has its own limitations related to cost, suitability and sustainability in resource-limited sites.

Why is DBS important for pediatric diagnosis?Easier to get blood samples by heel stick than venipuncture.

Ease of sample collection, storage and shipping. Testing can be performed in well-qualified central laboratories.

Problems with DBSSmall volume: about 50 - 100 ul per DBS spot

Extraction of nucleic acid from the blood card is labor-intensive and automation of the process is technically challenging

Presence of PCR Inhibitors

Current PCR based methodsDeVange Panteleeff et al; Rapid method for screening dried blood samples on filter paper for HIV-1 DNA. J.Clin.Microbiol., 37:350, 1999

Fisher et al; Simple DNA extraction method for DBS and comparison of two PCR assays for diagnosis of vertical HIV-1 transmission. J. Clin. Microbiol. 42:16, 2004

Problems: Detection sensitivity is low and thus requires nested amplification. These methods are not suitable for clinical settings

Use HIV total nucleic acid as the targets to increase the detection sensitivityWhen stored properly in humidity-free conditions, HIV RNA can be detected after several months.Storage conditions:humidity-tight bag desiccant packs and humidity indicator room temperature to -70C freezer

Storage of DBS

Chart1

11

0.16666666670.1232790881

0.080.0807720519

0.05263157890.0599540075

0.02375296910.0363979246

12 days

20 days

Storage conditions

Relative amount of HIV measured as compared with that of 23C dry conditions

010604-A(Yang, Gold enzymes)

010604-A(Yang, Gold enzymes)

YH did a couple DBS DNA tests along with K-1818. The results indicated that

the sensitivity was low. It turns out that she used 2002 Gold enzyme that I

gave her.

I did check the 2002 and 04 enzyme once on 122203 but with a strange

result. I ask her to repeat the experiment to make sure that the low detection

sensitivity is truly due to the enzyme before doing the next experiment.

No 2223-06 CD4=448, VL=476

No 3350-06 CD4=672, VL=2964

DBS and Provirus

Reagent25ul test8

10x PE2.524

MgCl2548Split the master mix into two tubes:

dNTP (+U)438.4A=87 ul + 1 ul Old enzyme (2002)

1624 PPP19.6B=87 ul + 1 ul new enzyme (2005)

1458E PPP19.6

Rox19.6Lot numbers:

UNG old0.32.882002B03271

02005E01533

water Q548

Total19.8190.08

5ul DNAAddressFam(Ad1)

1K-1818 1:2C329.65

2K-1818 1:8C431.79

3K-1818 1:32C534.24

4K-1818 1:128C634.7

5K-1818 1:2C730.73

6K-1818 1:8C832.66

7K-1818 1:32C934.23

8K-1818 1:128C1034.95

MX3000, 50(10")95(8')then 95(15")52(0.5') for 60 times RT= , started at

Dilutions of K-1818:

1:250 ul of K-1818 + 50 ul waterleft over = 95 ul; use 5 ul I tubes 1, 5

1:8above 1:2, 5 ul + 15 ul waterleft over = 15 ul; use 5 ul I tubes 2, 6



Results:The older enzyme works better than the new enzyme.

Next:We have a few tubes of old enzymes and thus she could use those to do a few

experiments.

DilutionsYr 2002Yr 2005

229.6530.73

831.7932.66

3234.2434.23

12834.734.95

010704-A(Yang, DBS ext)

010704-A(Yang, DBS ext)Extraction of DBS by Ou and Yang

DNA NoSerial NoByDateFilePCRHIVRNaseP

K-1572H-350DBS by Kim072903073003-A Ou10ul35.47630.389


Samples1H-350Done by H Yang

2H-351Done by H Yang

3H-350Done by Ou

4H-351Done by Ou

These samples were extracted by HY and Ou together.

1 DBS =>Use hole-puncher to get the DBS => 280 ul Lusis buffer (NP40,Tx100, Tris 8)

and 20ul proteinase => vortex 10 seconds =>60C 30 minutes, 500RPM

=>800 RPM 1' to remove foams=>Qiashredder, 14000RPM for 1 minute

=>Add 300ul AL to the shredder=>14000RPM for 1 minute

=>Add 300 ul 100% alconol, 5', 430 ul toa DNA column, spin 8000RPM for 1'

=>Repeat column loading once more

=> wash with 500ul AW-1, 8000RPM 1min then 500ul AW-2 8000RPM, 1min

=>14000 RPM 1 min, then elute the DNA using 55 ul water

Reagent25ul test10

10x PE2.527.5

MgCl2555

dNTP (+U)444

1624 PPP111

1458E PPP111

Rox111

UNG old0.33.3

Gold 20020.22.2

water Q555

Total20220

SampleVolwaterF(Ad1)Hex(Ad1)

1010704-D15ul52.2442.07

2010704-D25ul58.5843.75

3010704-D35ul6041.08

4010704-D45ul47.3740.54

5010704-D12.5ul2.5ul45.8938.69

6010704-D22.5ul2.5ul49.6541.76

7010704-D32.5ul2.5ul56.2240.96

8010704-D42.5ul2.5ul46.9439.33

9K-1818 1:25 ul37.8560

10water5 ul51.8860

MX4000, 50(10")95(8')then 95(15")52(0.5') for 60 times RT= , started at

Results:Very bad.

1. water control is positive (even is late is a very bad thing)

2. Positive control is way late, suggesting that the assay system is

not working right. Strange, we just checked the enzyme today and

concluded that it is working.

3. The extracted DNAs from the same DBS are not giving the same Ct

4. Lots of inhibitors (5ul's Cts are in general higher than those of 2.5ul)

Next:Check reagents. See next file (010704-B)

The reagents used in this experiment are HY's. She also used the gold from 2002.

(But this gold enzyme was examined yesterday and found to be very active).

010704-B(reagent check)

010704-B(reagent check)Reageent check

The DBS extraction experiment this morning done by YH and Ou was very bad. The

K-1818 control (1:2) had a Ct of 37.9. (expectation = 30). It is possible that

the reagents HY used were not quite right.

In this experiment, Ou uses his stock solutions and a few DNA to see if we could

get good results.

DNASampleDate

K-1572H-3500713003

K-1573H-3510713003

K-1863DBS cont

K-1864DBS cont

Reagent25ul test8

10x PE2.522

MgCl2544

dNTP (Roche*)435.2

1624 PPP18.837958

1458E PPP18.8K-18677744-06F936.23229.626

Rox18.8K-18687799-06F1039.92230.431

UNG old0.32.64

Gold 2005(Ou)0.21.76

water Q544

Total20176

*Roche 10mM total, and thus use 4 ul per 25ul assay

Sample 5ulDescriptionwater

1K-15720713003

2K-15730713003

3K-1867DBS cont

4K-1868DBS cont

5010704-D1today's

6010704-D2today's

7K-1818HIV cont

8waterNeg cont

MX4000, 50(10")95(8')then 95(15")52(1) for 60 times RT= , started at

010804-B(reagent check)

010804-B(reagent check)Reageent check

The DBS extraction experiment this morning done by YH and Ou was very bad. The

K-1818 control (1:2) had a Ct of 37.9. (expectation = 30). It is possible that

the reagents HY used were not quite right.

In this experiment, Ou uses his stock solutions and a few DNA to see if we could

get good results.

DNASampleDate

K-1572H-3500713003

K-1573H-3510713003

K-1863DBS cont

K-1864DBS cont

Yang's25ul test4Ou's25ul test4

10x PE2.51110x PE2.511

MgCl2522MgCl2522

dNTP (Roche*)417.6dNTP (Roche*)417.6

1624 PPP14.41624 PPP14.4

1458E PPP14.41458E PPP14.4

Rox14.4Rox14.4

UNG old0.31.32UNG old0.31.32

Gold 20020.20.88Gold 2005(Ou)0.20.88

water Q522water Q522

Total2088Total2088

*Roche 10mM total, and thus use 4 ul per 25ul assay

Sample 5ulDescriptionwater

1010704-D1

2010704-D2

3K-1818

4water

5010704-D1

6010704-D2

7K-1818

8water

MX4000, 50(10")95(8')then 95(15")52(1) for 60 times RT= , started at

010904-B

010904-B

Ou's25ul test8

10x PE2.522

MgCl2544

dNTP (Q)18.8Reagents are those Yang has today.

1624 PPP18.8She tossed out old ones that she had been

1458E PPP18.8using the last few days.

Rox18.8

UNG old0.32.64

Gold 2005(Ou)0.21.76

water Q870.4

Total20176

InstrumentAddressFam(3)Hex(3)F(Ad1)H(Ad1)

1K-1867C334.14626.874

21:27700C435.03428.02

31:4C535.33329.026

41:8C637.14429.977

5K-1868C738.15528.049

61:27700C836.80328.999

71:4C940.7230.013

81:8C106031.152

9K-1867D325.03

101:2MX4000D426.36

111:4D527.38

121:8D628.34

13K-1868D726.07

141:2MX4000D827.02

151:4D928.4

161:8D1028.93

50(10")95(8')then 95(15")52(1) for 60 times RT= , started at

7700K-1867 HexK-1868 HexMX4000K-1867 HexK-1868 Hex

126.87428.049134.14638.155

228.0228.999235.03436.803

429.02630.013435.33340.72

829.97731.152837.144

DilutionsMX K-1867 HexMX K-1868 HexABI K-1867 HexABI K-1868 Hex

125.0326.0726.87428.049

226.3627.0228.0228.999

427.3828.429.02630.013

828.3428.9329.97731.152

011204-A(MagaZorb)

011204-A(MagaZorb)Comaprison of two DNA extraction Methods

Whole blood samples:

1Inspire28590237991

2Inspire28602037993

120ul PK in a 2 ml tube (Cortex PK is stored at 4C, stable for 9 months)

2Add 200ul of whole blood

3Add 200ul of Cortex Lysis buffer, vortex 15 seconds

456C 10 minutes; 500 RPM

5Add 500ul Cortex binding buffer, 20ul beads (well-mixed)

6mix gently and incubate at RT for 10 minutes

7Put the tube onto a magnetic rack; Take out the sup after 30 seconds

8Add 1 ml of wash buffer, mix well by inversion

9Put the tube onto a magnetic rack; Take out the sup

10Repeat washing once more

11Use a Q-tip to remove excess washing solution, but do not touch the beads

12Add 100ul water, place the tube in a rocker, rocking at room temp for 10 minutes

13Place the tube onto a magnetic rack, take up the DNA containing water.

14Assign a serial number to the DNA.

Ou's25ul test12

10x PE2.533

MgCl2566

dNTP (Q)113.2

1624 PPP113.2

1458E PPP113.2

Rox113.2

UNG old0.33.96

Gold 2005(Ou)0.22.64

water Q8105.6

Total20264

SamplesAddressFam(3)Hex(3)F(Ad1)H(Ad1)

1Q-1C3

21:2C4

31:4C5

4Q-2C6

51:2C7

61:4C8

7M-1D3

81:2D4

91:4D5

10M-2D6

111:2D7

121:4D8

4000: 95(8')then 95(15")52(1) for 60 times RT= 2:26 , started at 4:55PM

Results:Quite good.

The results of Qiagen and Magnazorb are quite the same. No inhibitor detected

when 5 ul DNA is used.

Next:HY will start DBS DNA using the Megnazorb method. She will use the same two blood

samples for the DBS. See her notebook 011304.

Discussion:

Discuss with Debbie and HY separately in the AM.

HY spotted DBS at 9:30 AM and started DBS DNA isolation at ___ AM.

Ask Debbie to start preparing RNA work.

011304-A(DBS MagaZ)

011304-A(DBS MagaZ)

1Prepare DBS spots using the same Inspire specimens used 0n 01-12-04

(75 ul per circle x 8 for the first one and 10 for the second one)

2Use paper puncher to cut the filter papers into 2 ml tube

3Add 300 ul TE (10 mM Tris HCl, pH 8, 1 mM EDTA); Let stand at room temp

for 30 minutes with occasional mixing

4Add 20 ul of proteinase K (Cortex), followed by 200 ul lysis buffer (Cortex).

5Incubate at 56C for 10 minutes and agiate at 500RPM

6Transfer the content to a Qiagen shredder, change the collection tube .

with a new one having a cap. Spin at 14000 RPM for 1 minute

7Add 500 ul binding buffer (Cortex) and 20 ul of beads (well-mixed) to the DNA solution

8Incubate at room temp with constant shaking (use a rocker) for 10 minutes

9Place the tube on the Magnatic rack (Cortex) and remove the sup after 1 minute

with a fine-tip transfer pipette.

10Add 1 ml of washing buffer and gently invert the tubes (out of the magnetic rack) 20

times to disperse the beads. Place the tubes back to the magnetic rack and

take out the sup.

11Repeat the washing step once more

12Add 60 ul RNase and DNase free water. Mix in a rocker for 10 minutes.

13Sediment the beads and take out the DNA-containing water.

14Assign serial DNA numbers to the isolated DNA. These DNAs are ready for TaqMan detection.

Ou's25ul test10

10x PE2.527.5

MgCl2555

dNTP (Q)111

1624 PPP111

1458E PPP111

Rox111

UNG old0.33.3

Gold 2005(Ou)0.22.2

water Q333

Total15165

SamplesAddressFam(3)Hex(3)F(Ad1)H(Ad1)

1M-3C3

21:2C4

31:4C5

41:8C6

5M-4C7

61:2C8

71:4C9

81:8C10

9M-1C115ul

10M-2C125ul


011404-A(gold)

011404-A(gold)

Ou's25ul test8

10x PE2.524

MgCl2548

dNTP (Q)19.6

1624 PPP19.6

1458E PPP19.6A=88 ul

Rox19.6B=88 ul + 0.88 ul Gold

UNG old0.32.88

Gold 2005(Ou)0.21.92

water Q876.8

Total20192

5ulSourceAddressGoldF(Ad1)H(Ad1)H(Ad1)

1M-1DBSC3

2M-2DBSC41x

3M-3WholeBloodC5

4M-4WholeBloodC6

5M-1DBSC7

6M-2DBSC82x

7M-3WholeBloodC9

8M-4WholeBloodC10


Yang's record below

011404-A(gold)

Ou's25ul test8

10x PE2.5

MgCl25

dNTP (Q)1

1624 PPP1

1458E PPP1A=88 ul

Rox1B=88 ul + 0.88 ul Gold

UNG old0.3

Gold 2005(Ou)0.2

water Q8

Total20

5ulSourceAddressGoldFam(Adl3)Hex(Adl3)Fam(Adl)Hex((Adl)

1M-1DBS(HYK-1)C335.623033.4726.55

2M-2DBS(HYK-2)C41x38.4926.1835.6324.43

3M-3Whole BloodC5Na33.4545.2530.92

4M-4Whole BloodC638.0832.0335.6529.28

5M-1DBS(HYK-1)C735.926.0634.1624.18

6M-2DBS(HYK_2)C82x40.1827.2236.5324.95

7M-3Whole BloodC9Na28.22Na26.01

8M-4Whole BloodC1032.6726.7531.3624.96

MX4000: 95(8')then 95(15")52(1) for 60 times RT= 2:26 , started at 2:0PmPM

Results: written by OU

It appears that doubling the input of gold increase the detection sensitivity.

011304-A(DBS Magnaz)

1Prepare DBS spots using the same Inspire specimens used 0n 01-12-04

(75 ul per circle x 8 for the first one and 10 for the second one)

2Use paper puncher to cut the filter papers into 2 ml tube

3Add 300 ul TE (10 mM Tris HCl, pH 8, 1 mM EDTA); Let stand at room temp

for 30 minutes with occasional mixing

4Add 20 ul of proteinase K (Cortex), followed by 200 ul lysis buffer (Cortex).

5Incubate at 56C for 10 minutes and agiate at 500RPM

6Transfer the content to a Qiagen shredder, change the collection tube .

with a new one having a cap. Spin at 14000 RPM for 1 minute

7Add 500 ul binding buffer (Cortex) and 20 ul of beads (well-mixed) to the DNA solution

8Incubate at room temp with constant shaking (use a rocker) for 10 minutes

9Place the tube on the Magnatic rack (Cortex) and remove the sup after 1 minute

with a fine-tip transfer pipette.

10Add 1 ml of washing buffer and gently invert the tubes (out of the magnetic rack) 20

times to disperse the beads. Place the tubes back to the magnetic rack and

take out the sup.

11Repeat the washing step once more

12Add 60 ul RNase and DNase free water. Mix in a rocker for 10 minutes.

13Sediment the beads and take out the DNA-containing water.

14Assign serial DNA numbers to the isolated DNA. These DNAs are ready for TaqMan detection.

1132004

Ou's25ul test10

10x PE2.5

MgCl2555

dNTP (Q)111

1624 PPP111

1458E PPP111

Rox111

UNG old0.33.3

Gold 2005(Ou)0.22.2

water Q333

Total165

SamplesAddressFam(Adl3)Hex(Adl3)F(Ad1)H(Ad1)

1M-3C340.2632.6737.2830.4

21:2C4HYK-136.7828.735.1626.32

31:4C5285902Na27.38Na26.02

41:8C634.8230.6633.7628.03

5M-4C735.0429.6633.8127.41

61:2C8HYK-234.1527.7232.4125.64

71:4C928602035.522934.1426.75

81:8C1035.8930.1534.1427.47

9M-1C115ul34.4825.7533.0423.84

10M-2C125ul36.9528.4534.8125.69

MX4000: 95(8')then 95(15")52(1) for 60 times RT= 2:26 , started at 3:00PmPM

I find Gold is not good for adding, and I add another 2.00ul Gold to the same tube.

So the Gold is double.

Results: by Ou

It appears that doubling the gold input increases the sensitivity

DBS Summary(081303)

K-1489H-300DBS (100 ul)071503071503-B10ul38.09131.126

K-1602H-300DBS by Kim073003073003WL10ul35.8633.29

K-1490H-301DBS (100 ul)071503071503-B10ul38.95330.049

K-1507H-301DBS (100 ul) 3 step071603071603-A10ul6030.45

K-1511H-301DBS (100 ul) 2 step071603071603-A10ul35.37628.517

K-1603H-301DBS by Kim073003073003WL10ul36.5231.98

K-1491H-302DBS (100 ul)071503071503-B10ul39.7629.001

K-1492H-303DBS (100 ul)071503071503-B10ulExcluded

K-1493H-304DBS (100 ul)071503071503-B10ul37.76731.386

K-1604H-304DBS by Kim073003073003WL10ul35.5234.9

K-1494H-305DBS (100 ul)071503071503-B10ul37.76729.522

K-1605H-305DBS by Kim073003073003WL10ul34.932.58

K-1495H-306DBS (100 ul)071503071503-B10ul6060

K-1508H-306DBS (100 ul) 3 step071603071603-A10ul37.09328.312082303-A(Dif DBS)'!A1

K-1512H-306DBS (100 ul) 2 step071603071603-A10ul35.48127.307

K-1606H-306DBS by Kim073003073003WL10ul33.6930.84

K-1496H-307DBS (100 ul)071503071503-B10ul36.71831.041

K-1607H-307DBS by Kim073003073003WL10ul33.5231.33

K-1497H-308DBS (100 ul)071503071503-B10ul42.14632.239

K-1509H-308DBS (100 ul) 3 step071603071603-A10ul38.04227.47

K-1513H-308DBS (100 ul) 2 step071603071603-A10ul38.33228.291

K-1608H-308DBS by Kim073003073003WL10ul36.0132.06

K-1686H-308DBS by Ou081503081503-A10ul/5037.11131.428

K-1714H-308DBS by Ou082303082303-A10ul39.7432.63

K-1498H-309DBS (100 ul)071503071503-B10ul35.52529.419

K-1510H-309DBS (100 ul) 3 step071603071603-A10ul33.427.193

K-1514H-309DBS (100 ul) 2 step071603071603-A10ul34.0326.663

K-1609H-309DBS by Kim073003073003WL10ul34.1831.97

K-1515H-310DBS (100 ul) 2 step071703071903-A10ul36.86127.677072103-B10ul37.60728.332072303-A37.86730.613

K-1521H-310DBS 2 step072103072103-B10ulLost*

K-1531H-310DBS (100ul)072203

K-1535H-310DBS (100ul)072303072303-A10ul38.7128.676072303-A10ul38.14129.487

K-1516H-311DBS (100 ul) 2 step071703071903-A10ul41.36832.787072103-B10ul39.11532.027072303-A6034.467

K-1522H-311DBS 2 step072103072103-B10ul39.41232.025

K-1532H-311DBS (100ul)072203

K-1536H-311DBS (100ul)072303072303-A10ul38.15329.264072303-A10ul39.02929.25

K-1517H-312DBS (100 ul) 2 step071703071903-A10ul6031.208072103-B10ul6031.546

K-1523H-312DBS 2 step072103072103-B10ul6046.804

K-1533H-312DBS (100ul)072203

K-1537H-312DBS (100ul)072403072403-A10ul48.36728.025

K-1518H-313DBS (100 ul) 2 step071703071903-A10ul39.59429.178072103-B10ul37.6529.238

K-1524H-313DBS 2 step072103072103-B10ul44.7835.231

K-1534H-313DBS (100ul)072203

K-1538H-313DBS (100ul)072403072403-A10ul44.52630.706

K-1715H-313DBS by Ou082303082303-A10ul36.83729.202

K-1519H-314DBS (100 ul) 2 step071703071903-A10ul34.4927.416

K-1539H-314DBS (100ul)072403072403-A10ul38.1428.075

K-1520H-315DBS (100 ul) 2 step071703071903-A10ul41.14527.081

K-1540H-315DBS (100ul)072403072403-A10ul39.8826.848

K-1687H-315DBS (100ul)081503081503-A10ul/506030.676

K-1541H-316DBS (100ul)072403072403-A10ul35.68626.784

K-1542H-317DBS (100ul)072403072403-A10ul35.84227.938

K-1543H-318DBS (100ul)072403072403-A10ul39.19626.534

K-1544H-319DBS (100ul)072403072403-A10ul39.17827.189

K-1610H-320DBS by Kim073003073003WL10ul34.6632.68

K-1611H-321DBS by Kim073003073003WL10ul33.6730.87

K-1612H-322DBS by Kim073003073003WL10ul39.4533.81

K-1613H-323DBS by Kim073003073003WL10ul35.7532.56

K-1614H-324DBS by Kim073003073003WL10ul39.1531.13

K-1615H-325DBS by Kim073003073003WL10ul33.6333.55

K-1617H-326DBS by Kim073103073103 Ou10ul38.46730.921

K-1640H-326DBS by Ou080103080103-A Ou10ul7070

K-1618H-327DBS by Kim073103073103 Ou10ul7033.153

K-1641H-327DBS by Ou080103080103-A Ou10ul7033.27

K-1619H-328DBS by Kim073103073103 Ou10ul7037.192

K-1642H-328DBS by Ou080103080103-A Ou10ul7070

K-1665H-328DBS, 56(30)95(10)080703080703B-Ou5ul40.54227.592

K-1620H-329DBS by Kim073103073103 Ou10ul38.2534.163

K-1643H-329DBS by Ou080103080103-A Ou10ul37.58534.569

K-1621H-330DBS by Kim073103073103 Ou10ul42.14838.138

K-1644H-330DBS by Ou080103080103-A Ou10ul36.12132.017

K-1716H-330DBS by Ou082303082303-A10ul38.8231.724

K-1553H-331DBS by Wei072803072903-B Ou10ul41.83337.259

K-1645H-331DBS by Ou080103080103-A Ou10ul40.70434.464

K-1717H-331DBS by Ou082303082303-A10ul39.96527.397






K-1648H-336DBS by Ou AL separate080603080703-A-Ou5ul34.97531.676

K-1649H-336DBS by Ou AL separate080603080703-A-Ou5ul37.53237.719

K-1650H-336DBS by Ou AL to lysis b080603080603-B-Ou5ul35.67333.409

K-1651H-336DBS by Ou AL to lysis b080603080603-B-Ou5ul43.61460

K-1652H-336DBS080703Data not found

K-1653H-336Same as above but +AL080703Data not found

K-1718H-336DBS by Ou082303082303-A10ul36.61827.319



K-1688H-338DBS by Ou081503081503-A10ul/5047.48628.2690.0416666667

K-1719H-338DBS by Ou082303082303-A10ul41.85429.144





K-1565H-343DBS by Wei072903073003-A Ou10ul36.47632.874












K-1622H-354DBS by Kim073103073103-B10ul46.52339.065

K-1646H-354DBS by Ou080103080103-A Ou10ul37.73532.913

K-1720H-354DBS by Ou082303082303-A20ul37.16529.96





K-1616H-358DBS by Kim073003073003WL10ul37.0335.45



K-1689H-360DBS by Ou081503081503-A Ou10ul/5039.38231.161


K-1584H-362DBS by Kim072903073003-A Ou10ul7029.642

K-1623H-362DBS by Kim073103073103-B10ul7037.843

K-1647H-362DBS by Ou080103080103-A Ou10ul43.87134.432

K-1690H-362DBS by Ou081503081503-A Ou10ul/506030.073

K-1721H-362DBS by Ou082303082303-A13ul42.77728.759

K-1664H-377DBS, 56(30)95(10)080703080703B-Ou5ul35.23628.817

K-1693H-377DBS by Ou081503081503-A Ou10ul/5036.53230.139

K-1654H-378DBS, 56(30)95(10)080703080703B-Ou5ul37.4428.488

K-1655H-379DBS, 56(30)95(10)080703080703B-Ou5ul41.66226.244

K-1656H-380DBS, 56(30)95(10)080703080703B-Ou5ul33.59525.339

K-1657H-381DBS, 56(30)95(10)080703080703B-Ou5ul6028.222

K-1691H-381DBS by Ou081503081503-A Ou10ul/5043.6229.915

K-1658H-382DBS, 56(30)95(10)080703080703B-Ou5ul46.09227.582

K-1659H-383DBS, 56(30)95(10)080703080703B-Ou5ul34.69725.433

K-1660H-384DBS, 56(30)95(10)080703080703B-Ou5ul41.55329.551

K-1661H-385DBS, 56(30)95(10)080703080703B-Ou5ul38.05832.866

K-1662H-386DBS, 56(30)95(10)080703080703B-Ou5ul6035.651

K-1692H-386DBS by Ou081503081503-A Ou10ul/506029.175

K-1663H-387DBS, 56(30)95(10)080703080703B-Ou5ul35.88929.33

K-1666INSpire 279736DBS with gelatin-1080803080803B-Ou5ul37.06528.808

K-1667INSpire 279736DBS with gelatin-2080803080803B-Ou5ul44.16141.062

K-1668INSpire 279736DBS No gelatin-1080803080803B-Ou5ul37.34630.78

K-1669INSpire 279736DBS No gelatin-2080803080803B-Ou5ul37.43531.542

K-1597NH-101DBS by Kim073003073003WL10ul7030.35





011604(Yang)

011604(Yang)

H Yang tried 6 DBS samples (H-398 to H-402) yesterday afternoon but the

PCR results were very bad. Something in the extracted DNA causes the Rox

fluo progressively drop during PCR. HIV was only detected in 2 of the 6 samples

I can not tell if there is inhibitor in 10ul either because sometimes 10 is better

than 5 with 5ul giving no results

Thus, we should go back to recheck everything We'll use 4 samples previously

isolated by Wei with good results (072903) by WashU method.

072903-B(DBS=12)10ul in 25 ul assay

DNA 10ulSampleAddressFR3HR3

K-1561DBS H-339D337.54830.944

K-1562DBS H-340D435.44930.065

K-1563DBS H-341D538.15829.689

K-1564DBS H-342D638.04928.189

Ou's25ul test14

10x PE2.535

MgCl2570

dNTP (Q)114

1624 PPP114

1458E PPP114

Rox114Use 15 ul to tubes 1-4, then add

UNG old0.34.250 ul water to the master mix then

Gold 2005(Ou)0.22.8use 20ul to tubes 5-14.

water Q342

Total15210

DNAvolumeAddressF(Ad1)H(Ad1)H(Ad1)

1011604-D110C3

2011604-D210C4

3011604-D310C5

4011604-D410C6

5011604-D15C7

6011604-D25C8

7011604-D35C9

8011604-D45C10

9011504-D15D3

10011504-D25D4

11M-15D5

12K-18675D6

13washingB*5D7

14washingB*1D8


*washing buffer: from Cortex kit

01162004(4 DBS)

Yesterday the results were bad. Today Ou check the method which use 4 sample that

were done before. The samples are H-339, H-340, H-341, H-342.

Method:1 circle ==>200ul TE, Let stand at room temp for 30 minutes with

mixing. Add 20ulPK, then add 200ul lysis buffer. Incubate at 56C

for 10 minutes. Transfer the content to a Qiagen shredder and spin

at 14000RPM for 1 minute. Add 500ulbinding buffer and 20ul of beads.

Incubate at room temp for 10 minutes. Place the tube on the Magnatic

rack and remove the sup. Add 1ml of WB and do it again. Add 60ul

E-buffer and Mix. Sediment the beads and take out the DNA-containing

water.

DBS and Provirus

Reagent25ul test14

10x PE2.535

MgCl2570Use 15ul to tubes 1-4, then add 50ul

dNTP (Q)114water to the master mix then use 20ul

1624 PPP114to tube 5-13.

1458E PPP114

Rox114

UNG old0.34.2

Gold0.22.8

water Q342

Total15210

Sample IDDNA volumeAddressFam(Ad1)HEX(Adl)

1H-339HYK-910ulC344.2625

2H-340HYK-1010ulC441.3340.16

3H-341HYK-1110ulC54841.9

4H-342HYK-1210ulC646.0856.97

5H-339HYK-95ulC735.8628.39

6H-340HYK-105ulC833.9528.98

7H-341HYK-115ulC9Na29.34

8H-342HYK-125ulC10Na30.7

9H-389HYK-35ulD338.7932.96

10H-390HYK-45ulD432.9833.14

11285902HYK-15ulD5Na30.32

12K-1867K-18675ulD632.6222.86

13washingB*washingB*5ulD7NaNa

MX4000: 95(8')then 95(15")52(1) for 60 times RT= 2:26 , started at 1:00PM

Result: I Still do not understant the results.

011504(Yang)

011504(Yang)

011504(6 DBS)

The results of 011404 indicated that the Magnazorb extraction gave good

DNA recovery but there was still some PCR inhibitor present in the extract.

We decide to go ahead to use this method to extract DBS DNA. In this experiment,

we use 6 DBS that were checked previously with in-house modified Qiagen method.

SamplesHYK-

1H-3893

2H-3904

3H-3915

4H-3926

5H-3937

6H-3948

Method:1 circle ==> 200ul TE =====

DBS and Provirus

Reagent25ul test20

10x PE2.550

MgCl25100

dNTP (Q)120

1624 PPP120

1458E PPP120

Rox120

UNG old0.36

Gold0.24

water Q360

Total300


110ulC3NaNa

210ulC4NaNa

310ulC5NaNa

410ulC649.22Na

510ulC750.42Na

610ulC8NaNa

710ulC937.7131.97

810ulC10Na34.19

95ulD343.0731.29

105ulD4Na32.29

115ulD5Na31.03

125ulD6Na33.08

135ulD7Na32.83

145ulD8Na31.94

155ulD9Na31.6

165ulD10Na34.81

17K-186710ulE332.1826.77

18K-18675ulE431.8226.14


01162004(4 DBS)

Yesterday the results were bad. Today Ou check the method which use 4 sample th

DBS and Provirus

Reagent25ul test14

10x PE2.5

MgCl2570Use 15ul to tubes 1-4, then add 50ul

dNTP (Q)114water to the master mix then use 20ul

1624 PPP114to tube 5-13.

1458E PPP114

Rox114

UNG old0.34.2

Gold0.22.8

water Q342

Total15210


1HYK-910ulC344.2625

2HYK-1010ulC441.3340.16

3HYK-1110ulC54841.9

4HYK-1210ulC646.0856.97

5HYK-910ulC735.8628.39

6HYK-1010ulC833.9528.98

7HYK-1110ulC9Na29.34

8HYK-1210ulC10Na30.7

9HYK-35ulD338.7932.96

10HYK-45ulD432.9833.14

11HYK-15ulD5Na30.32

12K-18675ulD632.6222.86

13washingB*5ulD7NaNa


SamplesHYK-

1H-3893

2H-3904

3H-3915

4H-3926

5H-3937

6H-3948

Method:1 circle ==>200ul TE, Let stand at room temp for 30 minutes with

mixing. Add 20ulPK, then add 200ul lysis buffer. Incubate at 56C

for 10 minutes. Transfer the content to a Qiagen shredder and spin

at 14000RPM for 1 minute. Add 500ulbinding buffer and 20ul of beads..

Incubate at room temp for 10 minutes. Place the tube on the Magnatic

rack and remove the sup. Add 1ml of WB and do it again. Add 60ul

water and Mix. Sediment the beads and take out the DNA-containing

water.

DBS and Provirus

Reagent25ul test20

10x PE2.550

MgCl25100

dNTP (Q)120

1624 PPP120

1458E PPP120

Rox120

UNG old0.36

Gold0.24

water Q360

Total15300


110ulC3NaNa

210ulC4NaNa

310ulC5NaNa

410ulC649.22Na

510ulC750.42Na

610ulC8NaNa

710ulC937.7131.97

810ulC10Na34.19

95ulD343.0731.29

105ulD4Na32.29

115ulD5Na31.03

125ulD6Na33.08

135ulD7Na32.83

145ulD8Na31.94

155ulD9Na31.6

165ulD10Na34.81

17K-186710ulE332.1826.77

18K-18675ulE431.8226.14


Results: I do not understand the results.

012004-A(Isopropanol)

012004-A(Isopropanol)

We have a lot of small problems with DBS DNS isolation. The Qiagen

method gave a sensitivity of about 90% (could be 95%, but not reproducible)

and the new Cortex magnetic method also has inhibitors with the concentration

about the same observed in Qiagen's method. Bharat suggests to use isopropanol

ppt and alcohol wash to directly precipitate DNA. I am highly doubtful that

this will work but he thinks that it will.

In this test, I'll use Roche's male DNA to test the isopropanol ppt process.

10ul Male DNA ===> 1;2 dilution in 1x TE ====> 10ul 1:200 pGreen

DNAfinalDilution1' incubation

1dil 224359

2dil 446893

3dil 883977

4dil 16162107

5dil 32321312

6dil 6464641

7dil 128128411

8dil 256256290

DNA1x TECortexLBIsopropanolRT14KRPM70%AlcoholVortex14KRPMremove alcCortex EB

110ul200ulnone200ul10 min10 min1ml5 sec10 minair dry60

210ul200ulnone200ul10 min10 min1ml5 sec10 minair dry60

310ul120ul80ul200ul10 min10 min1ml5 sec10 minair dry60

410ul120ul80ul200ul10 min10 min1ml5 sec10 minair dry60

pG readings:

Control = 10ul Male DNA + 50ul cortex elution buffer

1:200 pG515 reading after 1 min

110 ul control diluted DNA10 ul3550

22 x dilution in TE10 ul2247



5TE only10 ul116

6Eluted DNA 1, 10ul10 ul85No DNA recovered

7Eluted DNA 1, 10ul, 1:2 in TE10 ul109No DNA recovered










Results:I did not get any DNA recovered this way.

Next:Check with Bharat to see if he has any ideas.

Use DBS to do this part.

Talked to Bharat and Xierong but no good conclusions

Sheet2

01202004(Method Check)Yang

I test 2 samples using MagaZorb method.

Method: 1circle ==>300 ul TE, stand at temp 30 minutes with

mixing. Add 20ul and 200ul lysis buffer, then at 56C for 10 minutes.

Transfer the content to Qiagen shredder . Add 500ul binding buffer

and 20ul of beads and at room temp for 10 minutes.Place the tube

on the Magnatic rack and remove the sup. Add 1ml of WB and do it

again. Add 60ul water amd mix. Sediment the beads and take out

the DNA-containing water.

DBS and Provirus

Reagent25ul test10

10x PE2.5

MgCl2550Tuble 1-4 are 10ul DNA volum. Tuble 5-9 are

dNTP (Q)1105ul DNA volum + 5ul water.

1624 PPP110

1458E PPP110

Rox110

UNG old0.33

Gold0.22

water Q330

Total150

Sample IDHYK-DNA volumeAddressFam(Ad1)HEX(Adl)

1285902110ulC340.6535.7

2285902110ulC4Na35.24

3286020210ulC538.3234.45

4286020210ulC642.6835.85

528590215ulC736.8527.64

628590215ulC837.3828.1

728602025ulC93427.59

828602025ulC1033.5727.52

9K-18675ulD333.9627.93


Result:

Reagent50 ul test11

10x PE555

MgCl210110

dNTP (Q)222

1624 PPP222Use 40ul to tubes 1-4160

1458E PPP222Use 20ul to tubes 9-1280

Rox222

UNG old0.66.6Left over (160 ul) + 1.6 ul Gold ==> use 40 ul

Gold0.44.4in tubes 5-8

water Q16176

Total40440

SamplesWashingsDNA volumeAddressFam(Ad1)HEX(Adl)

11x10ulC3

22x10ulC4

33x10ulC5

44x10ulC6

51x10ulC7

62x10ulC8

73x10ulC9

84x10ulC10

91x5ulD3

102x5ulD4

113x5ulD5

124x5ulD6

012104-A(SpecialCase)

012104-A(SpecialCase)

Sample ID: 286150

Requesting Investigator: Fay Cowart/Steve McDougal

Date sent from Lawenceville, 012004 and received the same day

Processing after receiving the sample: Being split to two vial but not equally

Then Zilma pooled them back together and realiquot into 5 vials.

I got one and ChunFu got one and the remaining two were stored

by Fay/Zilma in deep freezer. (01-21-04)

DNA processing:

DNA is to be isolated with Qiagen's QiaAmp by Ou today.

K-

Samples:1286150K-1871

2Inspire (16 day-old sample, stored at 4C since 1-5-04)K-1872

3NH-181 (stored at -20 by Wei)K-1873

Procedure:

200ul PBMC or blood + 20ul protease (Roche, Wei) 200ul AL=>56C 10 min

=> 200ul abs alcohol ==> QiaAmp column ==> AW-1 and AW-2 500ul wash

=> eluted in 55 ul water

DBS and Provirus

Reagent25ul test6

10x PE530

MgCl21060

dNTP Roche848

1624 PPP212

1458E PPP212

Rox 5uM212

UNG old0.63.6

gold0.42.4

water Q1060

Total40240

MX3000P: 50(10")95(8')then 95(15")52(1') for 60 times RT= , started at

10ulAddressPurposeResults

1K-1871C3unknownPositive

2K-1872C4(+)contrPositive

3K-1873C5(-)contrNegative

4waterC6waterNegative

5K-1867 5ulC7PCR(+)contPositive

6waterC8waterNegative

Rresults:

K-1871 is positive on proviral LTR DNA. All positive and negative controls

work out as expected.

However, K-1871's Ct is relatively high. This suggests that the proviral DNA

load is not very high. I should try on gag PPP too.

Also, I should use check HIV with or without 1624PPP. Hope that the Ct of HIV will

be lower (thus better).

Visit DateAgeDNA PCRRNALab*Comment

378801 daypositiveUMMC

3788910 days841FQ->841FQ->841FQ->22 to tubes 2,3,4

water Q566C= 21ul to tube 5

Total20264D= 92.4ul + 4.4ul 1286 then use 22 ul to tubes 6-9

tubesFProbe1Probe2Probe3RLocations

1128612931287int-A

2128412851283int-B

312848411283

4128412858411283

512918411283int-C

6128612851283int-D

712868411283

8128612931283

91286129312858411283

10133213311333gag

11LTR PPPLTR

12*128612881287/1283int-A

MX3000P95(8') 52(1') 60 times

*since there was enough leftover after 1283 was added, I used this leftover

and add 1286,1287 and 1288. This tube should be id to tube 1 with the exception

than 1293 was replaced with 1288. 1288 actually contains the sequence of 1293

plus about 15 nt at its 3'end

Results:Very infomative. I am glad to see these results.

LTR is still by far better than Roche's gag. And all the Int tested.

The int-C is looking good.

I think I could modify intC a little bit more to make it better:

First 841FT has to be changed to FQ or HQ

I'd like to see if the combination of LTR and Int-C will work.

The 841 probing region has some heterogeneity among HIV-1s, especially in subtype C.

Int-D: 3 prbes combined gave higher Rn but Ct does not change

Next:Can I pool LTR, gag and int together? OR just LTR and Int-C?

I do not like the idea of pooling too many Fam probes together. The baseline is going

to increase.

013104-B(Int-C optim)

013104-B(Int-C optim)Optimize Int-C

I would like to increase the DBS sensitivity by using two regions. The

Roche gag is not a good candidate because it is not very strong (sensitive).

In contrast, Int-C region (1291/1283, with 841 probe looks good). It is

only 2 Ct behind LTR. LTR has two copies/genome, and thus it is only 1Ct late.

If I could use more reverse primer and increase the probe concentration

like I did to LTR, then the gap can be narrowed. I like to get this done quickly

so that it can be used with the DBS project.

Reagent25ul test660C57C63C92bp

10x PE2.5181291->841FQ-> RT for 10 minutes==> use 5 ul for PCR

B=40ul+ 5ul DNase I==> RT for 10 minutes==> use 5 ul for PCR

DBS and Provirus

Reagent25ul test8

10x PE2.522

MgCl2544

dNTP Q18.8

1624 PPP18.8

1458E PPP18.8

Rox18.8

UNG old0.32.64

gold0.21.76

water Q326.4

Total15132

DNA 10ulRDD(Dnase buffer)DnaseHIVRNasePFam dRnH dRn

1K-94 1:50yesno29.9634.040.390.14

2K-94 1:50yesyes6060

3K-1867yesno35.3325.060.20.065

4K-1867yesyes6060

5031304B-R1nononega31.520.5

6031304B-R2nononega31.710.3

7031304B-R3nononega31.210.45

8031304B-R4nononega28.230.25

9K-94 1:50*nono23.5423.390.15

50(8)95(8')then 95(15")52(1') for 60 times RT= , started at

* unlike in tubes 1 and 2, this one is straight from the stock (1:50), no RDD

buffer was added. (thus the effect of RDD will not be observed in this tube)

Results:Dnase-I appears to work fine. But the buffer would change the background fluo

of HIV probe (not the Hex probe) to a much higher level.

Thus washing to remove DNase buffer is necessary.

031304-B sample does not seem to have much HIV DNA. (this is RNA DBS panel, thus

I am glad to see the absence of HIV DNA. The RNaseP is still there as expected.)

R1 in the panel has 1 million copies per ml of blood. It is DNA negative! This

means the MagaZorb RNA isolation is doing a great job in getting rid of DNA.

We may not need to worry about DNase treatment at all.

Thus overall, this is an experiment with good results.

I know now that DNaseI works well, and the RNA panel contains no HIV DNA.

031506-E(WB, 8E5 panel)

031606-A(RNaseP PPP)

031504-D(CDC-1 re-construction)

Xerong is going to start the revision of VL assay today. Great!

1790CDC-1F30gatcctgactgtacagccaggatccgtagt

1791CDC-1R21ccagcgtttaaagttattgtt

Starting DNA material: P-700

031703-A(WB std)

031506-E(WB, 8E5 panel)

2x dilution (with TB)

CountCounts

1102Need 13ml of blood, but I do not have that much

2188from a blood donor and thus I pooled two

3189persons' blood together

4140

5158

Average=155Dead cells about 10%

Cell numbers: I used plastic disposable counters:

and count 4 small squares (0.1ul per square)

3.9

Thus cell count = 155/4* 10000 * 2

=0.78 x 10^6 per ml

New nameper mlDilutionHow to diluteTotal madeExp CtStored wb (200ul)

WB-031504-14000020200ul culture + 3.8 ml blood3ml30.814 tubes

WB-031504-21000041 ml above + 3 ml blood3ml33.8414 tubes



WhoBlood std

Panel-2-1, 200ul

03-15-04 40K/ml

Results:14 sets made (one of them did not have tube 4). Store at -20 in 3235 for now.

Will give them to Wei tomorrow and she will start doing DNA isolation

using MagNaPure DNA kit for 233 samples from UNC. For each run, we

will have 4 positive controls. It will take 11 runs to finish them all.

For PCR, I'll titrate a set of PPPs (HIV-1 and RNaseP) this week

I need to get them done tomorrow so that they will be validated and Wei

will have time to test them once before I take my vacation next week.

031704-B(WB 8E5 panel)

031606-A(RNaseP PPP)

1624-C

ori oligoul

14604.88

146118.4total = 2 ml (water)

157357.68 vials

Tested on: see 021204 protocol021204 file

also HIV-1 PPP: 1458-I

I made 2ml but after adding the forward and probe, I realized that

there was not enough 1215 (Click

Qia(20/50, 03/09/04)Qia(20/50, 03/16/04)Mag(10/25, 04/13/04)Mag(10/25, 04/14/04)

IDFamHEXIDFamHEXIDFamHEXIDFamHEX

16022.3126022.73332.27830.524

440.0923.11538.0522.31

740.0422.8486022.21936.331.557

103922.63116022.681236.331.357

1340.3422.361436.9922.49

1637.322.71737.223.011828.6730.32

1938.01232037.7222.642129.328.49

2238.5523.072341.0523

2537.2823.212637.2222.152730.9628.89

2837.1922.32940.4823.22

3140.5323326023.723333.09129.629

346022.67356022.92366029.265

3736.4923.053837.2422.053931.8630

406022.47416022.454233.16528.719

Qia(20/50, 03/12/04)Qia(20/50, 03/17/04)

FamHEXFamHEX

4337.9623.194438.122.434532.81629.266

4638.5823.534739.1622.164833.56228.514

496022.81506023.015140.70429.555

526023.295339.2322.125436.830.006

5539.6823.175640.3622.13

5838.6923.335940.0122.52

616023.35626022.59636029.361

6439.9423.216537.923.03

6738.923.456837.7221.62

706023.34716023.04726029.273

7337.4423.037438.3123.2

7641.822.487737.5722.97

7938.7624.08806022.678132.34428.776

8238.2622.698341.3822.92

Update: As of 4-14-04 6PM

With the exception of 11 DBS samples, all the 3 circles of the 28 unique samples were used for

(1) Qiagen DNA; (2) Qiagen DNA again; and (3) MagaZorb total NA.

The results of two Qiagen DNAs are not identical. Four samples positive the first time were negative the second time and

One negative the first time was positve the second time. Six samples are negative both times.

I tried once to do nasted PCR but did not help. All negative samples were still negative with Nasted PCR.

Recently, we realized that even with 8E5 cells, we can use RTPCR to enhance the detection. Thus we use MagaZ (DNA beads)

to isolate total nucleic acids and ran them on RT-PCR. (see 04-13 and 04-14 results). We tested 17 of the 28 samples. The

results appear to be great (positive and negative controls were good). See table below for the comparison of these 17 samples

processed by DNA PCR and RT PCR. Four samples with positive Qiagen-1 and positive Qiagen-2 results gave the lowest HIV Ct

(28.67 to 31.86, with an average dCt of 7.1). The reduction of Ct in these 4 samples clearly shows that the power of

using MagaZ/RTPCR for DBS. The other 2 doubly positive samples had dCt of 5.1 and 5.0. Average dCt of all 6

Complilation of DNA results:

Negatives: 1,34,40,49,61 and 70 (n=6)

RNA/DNA results:

Negatives: 34,61 and 70 (n=3)

The difference between the two tests (Qia DNA and MagaZ RT) is 3 (1,40 and 49).

Is there a way to bring HIV DNA out of these 3 samples?

Or, we should not spend too much effort on these samples with low copy numbers.

IDQia-1Qia-2MagaZdCt

1637.337.228.678.5

1938.0137.7229.38.4

2537.2837.2230.966.3

3736.4937.2431.865.4

1606032.278

7938.766032.3446.4

4337.9638.132.8165.1

3140.536033.0917.4

40606033.165

4638.5839.1633.5625.0

10396036.32.7

740.046036.33.7

526039.2336.82.4

49606040.704

34606060

61606060

70606060

n=17

Report back to VQA

IDHIV detection

1Negative

4positive

7positive

10positive

13positive

16positive

19positive

22positive

25positive

28positive

31positive

34Negative

37positive

40Negative

43positive

46positive

49Negative

52positive

55positive

58positive

61Negative

64positive

67positive

70Negative

73positive

76positive

79positive

82positive

Click

041704-B(DBS Panel)

VQA Laboratory041504-A(Proficiency Report)

DNA Blood Spot Sensitivity PanelOu: we only report DNA results based on the

Shipped: Monday, February 09, 2004first Qiagen isolation and assay. (we modified

Due: Friday, April 16, 2004one neg to positive)

Lab Name:CDC (Chin-Yih Ou)

Lab Number:

Assay Name:Real-Time HIV-1

Brief Description of Assay Used:TaqMan-based assay (dual PCR; a cellular DNA is used as the internal control to monitor the DNA recovery and amplification efficiency)Extraction cost per sample:2.75

Type of Assay (Qualitative or Quantitative)Qualitative (semi-quantitative)Amplification and Detection cost per sample:2.25

Extraction Method:Qiagen DNATech Time per sample:Extraction: 1.5 hr; Realtime:2.5 hr

Amount of Spot Extracted:1 spot in 50ul water

Amount of Extract Amplified:20ul

Panel IDDate of ExtractionDate of AmplificationQualitative ResultQuantitative Result

004bs.013/9/043/9/04Negative

004bs.02

004bs.03

004bs.043/9/043/9/04Positive

004bs.05

004bs.06

004bs.073/9/043/9/04Positive

004bs.08

004bs.09

004bs.103/9/043/9/04Positive

004bs.11

004bs.12

004bs.133/9/043/9/04Positive

004bs.14

004bs.15

004bs.163/9/043/9/04Positive

004bs.17

004bs.18

004bs.193/9/043/9/04Positive

004bs.20

004bs.21

004bs.223/9/043/9/04Positive

004bs.23

004bs.24

004bs.253/9/043/9/04Positive

004bs.26

004bs.27

004bs.283/9/043/9/04Positive

004bs.29

004bs.30

004bs.313/9/043/9/04Positive

004bs.32

004bs.33

004bs.343/9/043/9/04Negative

004bs.35

004bs.36

004bs.373/9/043/9/04Positive

004bs.38

004bs.39

004bs.403/9/043/9/04Negative

004bs.41

004bs.42

004bs.433/12/043/12/04Positive

004bs.44

004bs.45

004bs.463/12/043/12/04Positive

004bs.47

004bs.48

004bs.493/12/043/12/04Negative

004bs.50

004bs.51

004bs.523/12/043/12/04Positive

004bs.53

004bs.54

004bs.553/12/043/12/04Positive

004bs.56

004bs.57

004bs.583/12/043/12/04Positive

004bs.59

004bs.60

004bs.613/12/043/12/04Negative

004bs.62

004bs.63

004bs.643/12/043/12/04Positive

004bs.65

004bs.66

004bs.673/12/043/12/04Positive

004bs.68

004bs.69

004bs.703/12/043/12/04Negative

004bs.71

004bs.72

004bs.733/12/043/12/04Positive

004bs.74

004bs.75

004bs.763/12/043/12/04Positive

004bs.77

004bs.78

004bs.793/12/043/12/04Positive

004bs.80

004bs.81

004bs.823/12/043/12/04Positive

004bs.83

004bs.84

041704-C(TRec RNaseP)

041504-B(EC-1 plasma)

200 ul plasma x 4 tubes

100ul 1x PBS

20 ul proteinase K (Magazorb)

200 ul MagaZ DNA lysis buffer (with 10 ul EC-1 1:100, 1st prep)

70C for 20 minutes; then 90C for 20 minutes

500 ul DNA binding buffer and 10 ul DNA beads

RT for 10 minutes

Put on Magnetic stand, leave for 2 minutes, remove the sup

Add 1 ml DNA washing buffer, turn the tubes 10 times, remove the sup

Repeat washing once more

Spin 6000 RPM, 1 minutes, put on Magnet stand, Use Q-tip to remove residual sup

Add 55 ul water, put on a rocker for 10 minutes

Put on Magnetic stand, leave for 2 minutes, recover the total NA.

Reagent25ul test8

5xQiagen Buffer544.0

dNTP Qia18.8

EnzymeMix Qiagen18.8ECRNA PPP:8

1458E PPP18.817900.43.52

ECRNA PPP2.219.417910.43.52

Rox18.8Probe18.8

RI (Roche)0.21.8total1.815.84

water Ou3.631.7

Total15132.0

RNA 10ulECEC sourceAddressFamHexHex

1today-B1FirstC36018.9781.56918

2today-B2FirstC46021.1291.63837

3today-B3FirstC56020.1691.64990

4today-B4FirstC66020.4451.82750

52FirstC76020.1011.611042

62FirstC86020.1091.81989

722nd, 100xC96018.7851.521045

822nd, 1000xC106023.1621.68900

MX4000: 50C(30)95(15')then 95(15")52(1') for 60 times RT= , started at

Results:MagaZ isolation of EC-1 appears to be not too bad. One of the 4 was low in Ct

(19) and the other 3 are pretty much the same.

2 ul of EC-1 gave consistently 20.1

However, I used too much 1791 (2x than needed)

041804-A(TRec RNaseP)

041504-C(HIV PPP)Making of HIV PPP

Use new reverse primer (1802)

1458I(2)

1458J

Testing of these probes:

Reagent25ul test741.8

10x PE2.519.25

MgCl2538.5

dNTP Qia17.7A=41.8 ul + 2.2ul 1458H

1624 PPP17.7B=41.8 ul + 2.2ul 1458I (2)

00C=41.8 ul + 2.2ul 1458J

Rox17.7

UNG old0.32.31

gold0.21.54

water Q861.6

Total19146.3

DNA 5ulAddressPPPFamHexdCtFamHex

1K-94 50xC3negativenegative

2K-94 500xC41458Gnegativenegative


4K-94 500xC61458I(2)negativenegative


6K-94 500xC81458Jnegativenegative

MX4000:95(8')then 95(15")52(1') for 60 times RT= , started at

Results:all negative. I make a mistake by not adding the 10x PE buffer

Next:Repeat. See 041604-A(HIV PPP check)

Result of next:

041904-A(TREC temp)

041604-A(HIV PPP)Making of HIV PPP

I made two new HIV-1 PPP (1458J and 1458I(2)) yesterday. I ran a PCR

yesterday but failed because of omitting PE buffer in the master mix

Here, I repeat the experiment again.

Testing of these probes:

Reagent25ul test741.8

10x PE2.519.25

MgCl2538.5

dNTP Qia17.7A=41.8 ul + 2.2ul 1458H

1624 PPP17.7B=41.8 ul + 2.2ul 1458I (2)

00C=41.8 ul + 2.2ul 1458J

Rox17.7

UNG old0.32.31

gold0.21.54

water Q861.6

Total19146.3

DNA 5ulAddressPPPFamFam FluodRn

1K-94 50xC328.1698201.963

2K-94 500xC41458G31.95101811.877

3K-94 50xC528.486971.597

4K-94 500xC61458I(2)32.3104381.871

5K-94 50xC728.27110131.874

6K-94 500xC81458J32.4184671.458

MX4000:95(8')then 95(15")52(1') for 60 times RT= , started at

Results:All three PPPs behave the same.

OK to use the two new ones.

041904-A(Steven-1)

041604-B(dNTP check)Checking of homemade dNTPs

Two new batches of dNTP made recently

dateconcbydUTPurpose

104-09-0410mM eachOuyesDNA

204-09-0410mM eachOuNoRT PCR

for comparison, use the following

310mM eachQiagenNoall

Reagent25ul test7

10x PE2.519.25

MgCl2538.5

0

1624 PPP17.7

1458H PPP17.7A=41.8 ul + 2.2 ul dNTP (dU) 4-9-04

Rox17.7B=41.8 ul + 2.2 ul dNTP (no dU) 4-9-04

UNG old0.32.31C=41.8 ul + 2.2 ul dNTP (Qia)

gold0.21.54

water Q861.6

Total19146.3

DNA 5ulAddressdNTPFamFlordRn

1K-94 50xC3Ou (+dU)29.58332915.518

2K-94 500xC436.24329475.234

3K-94 50xC5Ou (no dU)29.21325504.834

4K-94 500xC625.84317604.637

5K-94 50xC7Qia28.85307824.395

6K-94 500xC836.07318634.464

MX3000P(SS):95(10')then 95(15")52(1') for 60 times RT= , started at

Results:All three looked the same.

The two new ones can be officially used.

Note:4-20: dNTP (+dU) does not work at all with RTPCR. This was repeated two times

by Yang.. For real. Why?? I have no idea. Do not use this batch.

042004-A(Trec temp gradient)

041604-C(Trec)

New sets

1820TrecF21tttgtaaaggtgcccactcct

1821TrecR18tgcagggtttaggcacgg

1822=HCO-248F26tgccacatccctttcaaccatgctga

1823=HCO-249R26tattgcaactcgtgagaacggtgaat

1824TrecFP21FamQSY7cggtgatgcataggcacctgc

1825=HCO-224FP23FamQSY7cctaaaccctgcagctggcacgg

old sets:

HCO22330acacctttggtttttgtaaaggtgcccactFam/tamra

HCO22423cctaaaccctgcagctggcacggTet/tamra

HCO22520ctggcacgggccctgtctgcFam/tamra

HCO23121cacatccctttcaaccatgctHTFFrom Franco S

HCO23220tgcaactcgtgagaacggtgHTRFrom Franco S

HCO24826tgccacatccctttcaaccatgctgaFAdd 3 and 2 at the 5' and 3' of 231

HCO24926tattgcaactcgtgagaacggtgaatRAdd 3 and 3 at the 5' and 3' of 232

248->223 FT->join224 TT->225 FT->1820->joinjoin110.4RNaseP Conc

1823 5 uM0.45.76I=83.6 ul + 4.4 ul water

1462 HQ114.4II=83.6 ul + 4.4 ul 1847 5uM

1820 -> 5uM114.4III=83.6 ul + 4.4 ul 1847 (1:2 = 2.5uM)

1823 5 uM0.43.52

1847 5 uM0.45.8

18473-step

1462 HQ114.4B=83.6 ul + 4.4 ul Enzyme Mix->old way

MgCl2 25mM114.4C=83.6 ul + 4.4 ul Enzyme Mix->

UNG*0.34.3

water7.8112.3

total19273.6

*a new tube (not the same used yesterday);** mixture of Qia and Ou's 041104 prep.

Previous results, 060304-A =/- UNG

Std- 5ulFam CtHex CtFam dRHwx dRdCt

Std-133.5927.9732547775.62

Std-237.7727.7940829179.98

Std-341.0727.58366194913.49

Std-447.927.95320299719.95

First run, 060404-A, 3-step, 95(15")61(1)72(0.5)


1Std-128.1722.21679111295.96

2Std-231.7621.81597411569.95Slope=3.206

3Std-335.4221.795010117513.63eff = 100%

4Std-437.5222.15331131115.42(but one point can change the slope

MX400095(15' ); 95(15") and 61(1min)/72(30sec) for 50 times; started at 9:25AMeasily)

Second run, 060404-A, 2-step, 95(15")60(1)72(none)


5Std-127.6321.52559310016.11

6Std-231.1421.51500310259.63

7Std-334.8621.314810110013.55

8Std-438.721.284263124617.42

MX400095(15' ); 95(15") and 60(1min)/72(Zero sec) for 50 times; started at 11:55AM


9Std-128.123.3548110234.8

10Std-234.1423.944712103710.2

11Std-338.624.054596108714.55

12Std-438.3622.45188121715.96

13Std-544.4122.616779147621.8

MX400095(15' ); 95(30") and 61(1min)/72(Zero sec) for 60 times; started at 2:10PM

Results:

The best of the three protocols is with 2-step, 95(15"), 60C (1') and 72(none).

2-step, 62C is not better than 2-step 60C. Why? I do not know

But, as long as 2-step 60C works better, I'd go along with it.

060404A060404A060404A

log060304-A61/7560only62only

05.625.966.114.8

19.989.959.6310.2

213.4913.6313.5514.55

319.9515.4217.4215.96

421.8

060804-B(Yang, 1164 dil)

0000

0000

0000

0000

060304-A

61/75

60only

62only

060804-C(Yang, GAP on Std)

0

0

0

0

060804-D(GAP on CD8)

0

0

0

0

0

060904-A(Yang)

060404-B(H Yang's GAPDH check)

1st set of PPP11642nd set of PPP1167

11651168

1166 HQ1169 HQ

7

5x Buf542

dNTP Q18.4

enz mix18.4

Rox18.4Take out 17 ul out first and then

0add 1 ul 1458 PPP (HIV),2ul water

water975.6A=56.1ul + 3.3 ul each of 1164,65 and 66

total17142.8B=56.1ul + 3.3 ul each of 1167,68 and 69

*Inspire total NA isolated by Yang Hua today (060404)

InspRNA*ReagentAddressH(1)ndRnFluoHex

15ulAC3

21:2AC4

31:4AC5

45ulBC6

51:2BC7

61:4BC8

75ulHIV*C9

770095(15' ); 95(15") and 60(1min) for 50 times; started at

* serves as a positive control

061004-A(Yang)

060404-C(H Yang's GAPDH titration)

1st set of PPP11642nd set of PPP1167

11651168

1166 HQ1169 HQ

10

5x Buf557.5

dNTP Q111.5

enz mix111.5A=104.5ul + 5.5 ul 1168

Rox111.5B=104.5ul + 5.5 ul 1167

Insp total NA*557.5

1169 HQ111.5

water557.5

total19218.5

*Inspire total NA isolated by Yang Hua today (060404)

WATER1167(1:5)1168(1:5)Reagent1167Srength1168SrengthAddress

1410A=20ul0.2x1xC3

23.51.50A0.3x1xC4

3320A0.4x1xC5

42.52.50A0.5x1xC6

5050A1x1xC7

6401B=20ul1x0.2xD3

73.501.5B1x0.3xD4

8302B1x0.4xD5

92.502.5B1x0.5xD6

10005B1x1xD7

770095(15' ); 95(15") and 60(1min) for 50 times; started at

Preparation of 1167 (1:5) and 1168(1:5)

5ul of 5uM solution + 20 ul water = 25 ul total, 1:5

061004-B(Yang, efficiencies)

060404-D(Trec MgCl2)

Second run, 060404-A, 2-step, 95(15")60(1)72(none)


5Std-127.6321.526.11

6Std-231.1421.519.63

7Std-334.8621.3113.55

8Std-438.721.2817.42

MX400095(15' ); 95(15") and 60(1min)/72(Zero sec) for 50 times; started at 11:55AM

12

5x Buf572.0

dNTP Ou*114.4

Rox (LifeS)114.4

Trec PPP114.4

1460-> 5 uM0.45.8A=125.4ul + 8.8 ul water

1847 100 tubes

64

10K = 24, 10ul = 23

TREC Clone 8 Dilutions in Whole blood:dilution = 100x, Ct= 6.5

thus Ct = 30

But use 20 ul, thus Ct = 28

5ul

10K =24

Whole blood minus buffy coat

2 tubes of whole blood (10ml) =>Juan 2500RPM for 15 minutes

=> Remove the plasma carefully without touching the white cells

=> remove the while cells plus many red cells

'=>Add the above plasma back to the red cells.

=>these are whole blood minus white cells.

whole blood without whiteTrec 8 stockExpected Ct in 20ul

13 ml30 ul 10K diluted stock28

22.7 ml0.3 ml above31.3

32.7 ml0.3 ml above34.6

42.7 ml0.3 ml above37.9

52.7 ml0.3 ml above41.2

Aliquots for storage and DBS

0.2 ml aliquotstubes (0.2ml) storedDBS made

1TREC8 Std-A-11020

2TREC8 Std-A-21020

3TREC8 Std-A-31020

4TREC8 Std-A-41020

5TREC8 Std-A-51020

15 ml 8E5 old culture => spin in Eppendorf 7700 5' to collect 8E5 cells

==>added to 4 ml of de-WBC whole blood.

=>saved in 4C overnight. I could not work on it today.

I need to repeat all these next week.

061604-E(Heat on RT-PCR)

061404-A(Yang, Std curve)Construction of a HIV/GAPDH standard curve for Yang's

project

Yang Hua had calibrated the GAPDH PPP for the use of the HIV RNA quant in DBS.

Thus we now need to have a standard curve. To constuct it, we'll use negative

whole blood to dilute HIV and put them on DBS.

Before using DBS, we will do the dilution in whole blood and use them directly.

6

5x Buf533

dNTP Q16.6

enz mix16.6

11640.31.98

11650.31.98

116616.6

Rox(lifeSci)16.6

1458PPP16.6

water9.462.04

total20132

Dilutions of two GAPDH primers

HIV-1 positive plasma: From Ou's VL panel (5x10^7 per ml)

Negative whole blood: Freshly obtained this morning from LL

Dilution of HIV plasma:

Negative bloodPlasma*HIV/ml

10.8 ml88.8 ul undil plasma*5,000,000

40.8 mlabove 88.8 ul500,000

30.8 mlabove 88.8 ul50,000

40.8 mlabove 88.8 ul5,000

50.8 mlabove 88.8 ul500

* undiluted plasma = 5 x 10^7 per ml

them into ten vials.

The first tube represents a dilution of 10x from the plasma stock (which is 5 x 10^7/ml)

and thus the titer will become 5 x 10^6 /ml. This is good for infant work because

infants have high titers. (we may have to use a higher conc to begin with later to

cover 1x 10^7/ml since many infants have titer of that high titer).

1. Use 200ul diluted standards for RNA isolation directly.->Beads=>55ul=>use 5ul/RT PCR

2. Spot DBS (50ul per circle) and then use them for RNA isolation

HIV/mlHIV /testedF(1)H(1)dCtF dRH dR

15,000,000100,00031.925.16.84141361

2500,00010,00037.325.3123566392

350,0001,00039.0724.814.273841360

45,0001006025.140575

550010600571

600606000

Results:Only three points showed up. GAPDH Cts are consistent.

But I am not sure if the concentrations could be lowered a little bit more.

IF GAPDH PPP is lowered, the forth point may show.

BUT, most of the infant DBS has a Ct lower than 40.

IF the same blood with HIV is spotted onto DBS and extracted, we will

have a very high Ct. That was not what Debbie got Need to ask Debbie.

Whole blood resultsDBS expectations

HIV/mlHIV /testedF(1)H(1)F(1)H(1)

15,000,000100,00031.925.136.429.6

4500,00010,00037.325.341.829.8

350,0001,00039.0724.843.5729.3

45,0001006025.1460

5500106060

770050(30')95(15' ); 95(15") and 60(1min) for 60 times; started at

Overall, the detection of HIV-1 is vdery poor. The first data point was 5 million/ml

and gave a Ct of 32. Ct 32 is way too high for 5M/ml.

Next:Need to make sure the dilution of virus in whole blood and the extraction of RNA

and DNA were correctly done. DO this exp again and with whole blood and negative

plasma.

061604-E(Heat on RT-PCR)

0

0

0

dCt

061704-A(Heat on RT-PCR)

061404-B(CynthiaWarner)

Got a phone call from Cynthia Warner:

She could not get Roche Qualitative PCR reagent in Careb lab and thus

could not do pediatric testings.

She is thinking to use Roche viral load assay to work on infants' plasma

and uses it for infant HIV diagnostics.

Barbado lab has been doing VL, and they may have done pediatric VL for

about 10-20 samples.

She wants to have someone as external QC and I told her to use Tom Spira's

lab (because I am not doing VL myself, not CLIA certified).

She asked me if frozen plasma from infants was good. I said yes. But whole

blood in DBS is good because we not only detecting virus in the plasma, we

also detect HIV RNA and DNA inside the cells.

She mentioned Tom Hearn, Mark Rayfield and Marcia Kellish's involvement.

I am not sure what test Marcia has for infants. She does not quite know either.

As far as the training of Katherine Chang Kit goes, she does not really give

me a definate answer. She will continue working on it and may also ask another

person from Barbado to come too.. Her decision, I think, depends upon how her

communication goes with GAP. GAP people may be the one decides everything.

I think I just have to have patience. Anyway, I have the Uganda/Cameroon project

almost finished. I need to write the manuscript quickly and send it up (also

to GAP office).

061704-B(RNase on DBS)

061404-C(Pau's PPP)

SetpppHXB2Size

LTR-1 F1829521agcctcaataaagcttgccttgag=331 + 1 g at 3')

LTR-1 R183260585ggtctgagggatctctagttacc=almost = 1209

LTR-1 P1836 FQ

LTR-4 F1827412ccctcagatgctgcatataagcOu's comment:

LTR-4 R183150089tagccagagagctccc(a/g)ggThis set would only work on

LTR-4 P1835DNA, not on RNA

gag-1 F18291375gcagccatgcaaatgttaaaagaalmost =SK145', ND-1346; 433

gag-1 R18331481107ggttctctcat(c/t)tggcctggtgalmost =1349

gag-1 P1837 FQATCAATGAGGA(A/G)GCTGCAGAATGGalmost =1350

Int0 F18304650attccctacaatccccaaagtca(a/g)ggagThis site is different

Int0 R18344776127gaatgaatactgccat(c/t)tgtactgctgtfrom what I have been using

Int0 P1838 FQthus I can try it.

0

I need a gag and a integrase PPPP to confirm the 8 discordant samples in the Cameroon infant DBS

project. Pau kindly offer the above 4 PPPs. I do not need LTR PPPs. His gag PPP is essentially

the same as the Amplicor 1.5 PPP and several of my versions of PPP in the same area and thus

I may not need it at all. His Int0 PPP is on the other hand different from what I used and thus

may be helpful.

061504: Hua is preparing 20 samples (including the 8 discordant samples) today, and thus

I need to proceed with this work quickly.

I think I would use my own gag and int for now (since I know their performance myself well)

and use them for a few DBS samples first. If they worked out alright, I'll give them

to Hua to use.

061704-B(Heat on RT-PCR)

061405-A(Yang, Std curve)

Negative bloodNegative plasmaPlasma*HIV/ml

10.2 ml22.2 ul undil plasma*5,000,000

20.2 mlabove 22.2 ul500,000

30.2 mlabove 22.2 ul50,000

40.2 mlabove 22.2 ul5,000

50.2 ml22.2 ul undil plasma*

60.2 mlabove 22.2 ul



* undiluted plasma = 5 x 10^7 per ml

8

5x Buf544

dNTP Q18.8

enz mix18.8

11640.32.64

11650.32.64

116618.8

Rox(lifeSci)18.8

1458PPP18.8

water9.482.72

total20176

HIV/mlDilutentHIV /testedF(1)H(1)dCtH dR

15,000,000whole blood100,000

2500,000whole blood10,000

350,000whole blood1,000

45,000whole blood100

55,000,000plasma100,000

6500,000plasma10,000

750,000plasma1,000

85,000plasma100

770050(30')95(15' ); 95(15") and 60(1min) for 60 times; started at

061704-C(Trec DBS standards)

061504-B(4 HIV PPPs)

gag134713491350 FQ

Int-A837716841 FQ

Int-B1291765841 FQ aliq 3.5 ml per tube x 10 tubes, and 3 ml per tube x 2 tubes

=> spin in Jouan 2500 for 15 minuts

3.5 ml = 5 million cells.

U-Blood:Whole blood-minus white cells plus U937 cells

See 6-14-04 for the preparation of the whole blood-minus WBC cells.

See below, I need approximately 3 ml of U-Blood. Or 6 tubes of

pelleted U-937 cells in 3 ml of blood without white cells.

The other U-937 cells ===> stored at -20C

Preparation of U-Blood with TREC

tubesU-bloodTrec 10^5 stockYieldWhat for:Trec/assay*

10.6 ml30ul0.3 ml0.2 ml for Qiagen DNA500

20.3 ml0.3 ml of mixture above0.3 mland the remainder for250

30.3 ml0.3 ml of mixture above0.3 mla big DBS circle125

40.3 ml0.3 ml of mixture above0.3 mlThis is done for all 562

50.3 ml0.3 ml of mixture above0.6 mlsamples31

* if 200ul blood is isolated with QiaDNA, eluted in 100ul and 1 ul is used per assay

This panel needs 1.8 ml of U-Blood

Preparation of cell number panel

This panel is used to determine the cell number using RNaseP

tubesU-bloodBlood-WBCTrec 10^4 stockYieldWhat for:Cell #/4ul/assay

1200 ul06.25 ul206ul0.2 ml for Qiagen DNA40,000

2100 ul100 ul6.25206ulnothing for DBS20,000

350 ul150 ul6.25206ul10,000

425 ul175 ul6.25206ul5,000

Sheet10

061704-D(Heat on RT-PCR)

10

5x Buf557.5

dNTP Q111.5

enz mix111.5

VLPP-12111.5

0A=110 ul

Rox(lifeSci)111.5B=110ul + 70C for 5 minutes

water11126.5

total20230

5ulRT enzymeF CtH CtdCt

1YHM-01437.923070na

2YHM-015No 70C*34.5132596.36

3YHM-01632.3535918.63

4YHM-01728.9437206.19

5CDC-1 Phage*6019.96090323.28

6YHM-014602742

7YHM-01570C 5 min40.873039

8YHM-01640.983349

9YHM-01735.130

10CDC-1 Phage*6043.240715

MX3000P (SS)

*: 5ul CDC-1D + 45 ul water +>75C for 3 minutes, then use 5 ul each in tubes 5 and 10

Results:Very nice.

170C destroys RT activity completely (Ct of CDC-1 changes from 20 to 43).

270C treatment does not affect DNA (in another experiment yesterday,

061704-A(Heat on RT-PCR); thus we can use heat treatment to measure

DNA content directly.

3The amount of RNA and DNA in 8E5 cells:

TotalRNAdCt

YHM-01437.9260

YHM-01534.5140.876.36

YHM-01632.3540.988.63

YHM-01728.9435.136.19

average7.06

ratio=130

Thus there are about 130 times more RNA copies than DNA in 8E5 cells.

4How to measure relative RNA and DNA content in cells?

1Take 5 infant DBS total NA

2Measure total NA

3Measure DNA (heated RT PCR reagent)

4Calculate dCt and then derive the RNA/DNA Ratio

DNA copies/circleTotalDNA only

4037.92

20034.5140.87

100032.3540.98

1000028.9435.13

DNase treatment

0.4166666667

0.525

Sheet10

00

00

00

00

Total

DNA only

062404(Hua)

062104-A(Trec Protocol)

Protocol for the TREC detection in infant DBS

Specimens:

6 NH specimens; NH-151 to NH-156

UBT DBS standards: UBT-2, -3, -4, -5 and -6

DBS DNA isolation:

1Size of DNA: One 6mm punch per sample

2Isolation procedure: Use Cortex DNA kit

(see DBS DNA isolation protocol file)

DNA (actually total nucleic acids) is eluted in 55 ul

10 ul will be used in a 25 ul assay reaction.

It is possible that the amount of DNA in this DNA is not enough.

We may have to use 20 ul in a 50 ul reaction.

We need to evalute the 10ul assay.

1Add 300ul PBS and 20ul Proteinase K to each 6mm DBS punch in a 2ml tube

then add 200ul Cortex lysis buffer

270C (30min) then 90(20min); 1000 RPM;

spin 8000 for 1 min to remove condensations.

3Transfer the content of the tubes to a new clean 2 ml tube

containing 500ul of Binding buffer (DNA ) and 20ul Beads (DNA)

4

5Put the tubes on a magnetic rack. Stand for 2 minutes

6Remove the sup with a fine-tip pipette

7Add 1ml of Washing buffer (DNA or RNA)

8Put the tubes onto our automated washer, wash the beads 10 times

9Aspirate sup with a fine tip transfer pipette


11

12Remove the sup (about 25ul) with a Q-tip

13Add 55 ul water and put the rack on a rocker for 15 minutes

14Put the tube back to the Magnet and let stand for 2 min.

15Take out 50ul with a 200ul pipette tip

Assay:Use Qiagen's RT PCR reagent (25 ul per assay)

See notes above. 50 ul reaction may be needed.

8Trec PPP060204

5x Buffer544.01820F=200nMM

dNTP Qiagen18.81823400nM

Rox (LifeS)18.81824400uM

Trec PPP18.8RNaseP DNA PPP

EnzymeMix18.81460-> 5 uMF=80nM

1460-> 5 uM0.43.51847 ----1855FQ, 1856CQ ---> gi|17425231|dbj|AP001582.4|

Homo sapiens genomic DNA, chromosome 11q, clone:RP11-215H18, complete

sequence

Length = 90256

Query: 1 tctaccgtgcaagttcattatcgaa 25

|||||||||||||||||||||||||

Sbjct: 53410 tctaccgtgcaagttcattatcgaa 53386

Query: 26 tgtgccagagctgtgtggagctgg 49

||||||||||||||||||||||||

Sbjct: 52917 tgtgccagagctgtgtggagctgg 52894

cho2:For SCID work

cho2:same as 1856, for Fam first, if OK, uses Cy5 to work with Trec Fam probe

cho2:same as 1855, for Fam first, if OK, uses Cy5 to work with Trec Fam probe

>gi|17425231|dbj|AP001582.4|

071004(Hua PI)

070204-C(Vogt)More Texas DBS and cord blood from Alabama

So far we have checked 21 Texas newborn DBS using 4 3mm punch per DBS and all

21 samples were positive for Trec (great TREC Cts around 32-35 and RNaseP Ct

of 29-30).

Here we (Bob Vogt) will continue working on Texas DBS

DBS DNA isolation:

1Size of DNA: Four 3mm punch per sample

1Texas-22FS-055

2Texas-23FS-056#'062204-B(Vogt)'!A1

3Texas-24FS-057#'062504-A(Vogt)'!A1

4Alabama Cord-0114011FS-058#'062904-B(Vogt)'!A1

5Alabama Cord-0214014FS-059

6Alabama Cord-0314020FS-060

7J Puck Control 12-5-03FS-061Isolation of DNA was carried out

8J Puck Control 12-5-03FS-062by Bob.

9J Puck depletedFS-063

10J Puck depletedFS-064

2Isolation procedure: Use Cortex DNA kit

(see DBS DNA isolation protocol file)

DNA (actually total nucleic acids) is eluted in 55 ul

10 ul will be used in a 25 ul assay reaction.

1Add 300ul PBS and 20ul Proteinase K to each 6mm DBS punch in a 2ml tube

then add 200ul Cortex lysis buffer

270C (30min) then 90(20min); 1000 RPM;

spin 8000 for 1 min to remove condensations.

3Transfer the content of the tubes to a new clean 2 ml tube

containing 500ul of Binding buffer (DNA ) and 20ul Beads (DNA)

4

5Put the tubes on a magnetic rack. Stand for 2 minutes

6Remove the sup with a fine-tip pipette

7Add 1ml of Washing buffer (DNA or RNA)

8Put the tubes onto our automated washer, wash the beads 10 times

9Aspirate sup with a fine tip transfer pipette


11

12Remove the sup (about 25ul) with a Q-tip

13Add 55 ul water and put the rack on a rocker for 15 minutes

14Put the tube back to the Magnet and let stand for 2 min.

15Take out 50ul with a 200ul pipette tip

Assay:Use Qiagen's RT PCR reagent (25 ul per assay)

13

5x Buffer565.0

25mM MgCl2113.0

dNTP Qiagen113.0

Rox (LifeS)113.0

Trec PPP113.0

1852 PPP*113.0

EnzymeMix113.0

UNG0.33.9*: RNaseP PPP

water3.748.1

total15195.0

TubesAddress10ulDescriptionF(0.1)H(0.026)FH

1C3FS-055Texas-2242.1634.8250153387

2C4FS-056Texas-236035.2804240

3C5FS-057Texas-2440.8134.28306193959

4C6FS-058Alabama Cord-0141.0835.08233573864

5C7FS-059Alabama Cord-0241.7633.63311774987

6C8FS-060Alabama Cord-0343.2133.91306174835

7C9FS-061J Puck Control 12-5-03035.8306824

8C10FS-062J Puck Control 12-5-0337.228.48328484688

9D3FS-063J Puck depleted42.925.48288275145

10D4FS-064J Puck depleted38.5425.43319964626

11D5clone8 1:10KPositive control24.260317810

12D6waterNegative Control0000

MX3000P (SS)95(15') then 95(15") and 60(1') or 60xStarted at 4:40, 2:17

*:Use 2.5 ul DNA and 7.5ul water

Results:Not good. RNaseP Cts were too high form the Tx and AL samples. This indicates

that the isolation or the quality of the DNA was not good. Thus the TREC Cts

were high too. Need to repet this experiment.

Next:Take 2 samples from this experiment and two from a previous successful experiment

and run them to see if the problem is due to NA extraction. See 070404 (Ou).

cho2:received 070204 by Bob Vogt

cho2:received 070204 by Bob Vogt

#'062504-A(Vogt)'!A1

#'062204-B(Vogt)'!A1

#'062904-B(Vogt)'!A1

071004(Hua, Cam PI)

070204-D(CD8 in DBS)

#'060704-C(CD8mRNA)'!A1

According to the results of 060804-A,#'060804-A(CD8 mRNA 3PPPs)'!A1

the best CD8 mRNA PPP is 1056/1057/1058HQ#'060804-D(GAP on CD8)'!A1

1056FCD8-a,ex3/4gggcgcagtgcacacga

1057RCD8-a,ex4gtgacaggagaaggacccca

1058PCD8-a,ex4cgcccctggccgggacttHexQSY7

1850PCD8-a,ex4cgcccctggccgggacttFamQSY7

1058 should be replaced with 1850 since 1850 is fam labeled and is easier to detect

for now.

CD8 message is also present in NKs. Thus it is not as good as CD3.

CD4 mRNA PPPOu03-A.xls#'011203(1 CD4 PPP=200nM)'!A1

1712/13/09===>Ou03-A.xls#'121203-B(New CD4PPP)'!A1

GAPDH mRNA PPP===>Ou04-A (version 1) (version 1) (version 1).xls#'060704-B(GapDH; Yang)'!A1

1164

1165

1166 HQ

6

5x Buf536

dNTP Q17.2A= 37.4 ul + 2.2 ul each of1056;1057;1850

enz mix17.2B= 37.4 ul + 2.2 ul each of1712;1713;1709

Rox17.2C= 37.4 ul + 2.2 ul each of1164;1165;1166

water964.8

total17122.4

TubesAddress5ulDescriptionGeneF(0.1)H(0.026)FH

1C3FS-047Cameroon inf DBS31.56012260

2C4FS-048Cameroon inf DBSCD832.16021970

3C5FS-047Cameroon inf DBS060150

4C6FS-048Cameroon inf DBSCD40602060

5C7FS-047Cameroon inf DBS6029.06029.06

6C8FS-048Cameroon inf DBSGAPDH6028.54028.54

Results:Not good enough.

CD4 did not work at all, and in general, CD8 and GAPDH are both very weak.

Something was not quite right.

Maybe this DBS NA was not extracted well. But their DNA appeared to

be good (see 070204-A)

The good thing is, however, CD8 gave some signal.. I can continue working

on it until CD3 PPP is here.

When I used this CD8 PPP last time (060804)#'060704-C(CD8mRNA)'!A1

I did not get a great result either. The Ct from an Inspire total NA

was 32 and dR was 750 (Hex, not Fam)

Next:Check on this CD8 PPP more. The probe is new and is labeled with Fam. (the previous

one 1058 was labeled with Hex). In practice, Fam should gield a better result than

Hex, but I did not see that this time.

I need to increase the concentrations of the reverse primer and probe.

Also, I should use RNA isolated from a normal route (not from the small 250ul vials).

#'060704-C(CD8mRNA)'!A1

#'060804-A(CD8 mRNA 3PPPs)'!A1

#'060804-D(GAP on CD8)'!A1

Ou03-A.xls#'011203(1 CD4 PPP=200nM)'!A1

Ou03-A.xls#'121203-B(New CD4PPP)'!A1

Ou04-A (version 1) (version 1) (version 1).xls#'060704-B(GapDH; Yang)'!A1

#'060704-C(CD8mRNA)'!A1

071204-B(Hua Std PI)

070304-A(CD8mRNA)CD8 mRNA PPP titration

The results of CD8 mRNA detection on 070204-D was not good. (It may be good

but the Ct seems to be too high)

The new probe 1850 FQ may be off in its concentration. Thus in this experiment

I want to check this probe. I'll increase the reverse primer concentration

to 400nM too.

6

5x Buf533

dNTP Q16.6

enz mix16.6

Rox16.6

1056 5uM16.6

1057 5uM213.2

FS-027533 Buffy coat + non-buffy coat cells

===>Ficoll Hypaque ==> PBMC

Start Screening DBS samples?

Any samples with low CD3?

080504-A(Qia AVL)

072104-A(TREC vs CD3)

4

5x Buf524

dNTP Q14.8A=66ul + 4.4 ul 1867, 8.8 ul ea of 1869 and 1870

enz mix14.8B=66ul + 4.4 ul of TREC PPP

Rox0.20.96

1852-B*14.8

water6.832.64FS-065Texas-2234.8427.01

total1572FS-066Texas-2335.1627.54

*=RNaseP PPPFS-067Texas-2433.9227.78

FS-068Alabama Cord 1403135.7127.37

Address5ul 1:2FGeneFHFHdCtav

1C3FS-065Texas-2232.2830.532033729761.750.38

2C4FS-066Texas-23CD329.5430.39201932946-0.85

3C5FS-067Texas-2429.4230.65196523053-1.23

4C6FS-068Cord 1403131.2329.381974920101.85

5C7FS-065Texas-2235.930.453585827945.455.22

6C8FS-066Texas-23TREC36.3230.223496730286.1

7C9FS-067Texas-2434.5830.73607531203.88

8C10FS-068Cord 1403135.0929.633502328385.46

MX3000P (3270)50(30')95(15'), 95(15")60(1') for 60 timesdif=4.84

power=29x

Results:

As expected.

CD3 is about 5 cycles shorter than TREC (about 30x only).

I may want to change to another RT PCR system to see if I can get a better improvement.

Next:Order Dynal beads for CD15 (granulocytes), CD19 (B cells). (ordered by Bob Vogt)

CD15 beads will come tomorrow but 19 is back-ordered.

Use these beads to find out the background of CD3 mRNA in these major blood cells.

080504-B(Qia vs Cortex)

072104-B(CD3 Cy5)CD3 Cy5 probe

1856: 10uM

430ul probe + 95 ul water = 125ul

5x Buf522

dNTP Q14.4

enz mix14.4

Rox0.20.88

1867 5uM14.4

1870 10uM14.4

RNaeP PPP*14.4

FS-2214.4

water12.856.32

total24105.6

FamCy5

1869 10uM1856 10uMFCH

1128.066033.35

2127.796033.3

31606033.22

41606033.34

MX4000 (BP)50(30')95(15'), 95(15")60(1') for 60 times

Results:Cy5 did not work. Why, it is a probe for CD3 delta, not for CD3 zeta.

The primers are for zeta Stupid mistake.

Next:Order a CD5 probe for CD3 zeta.

080504-C(Bead temperature)

072104-C(CD3 detune)Detune of CD3 PPP

It may be useful to put CD3 mRNA and TREC DNA detection together for

a confirmatory test. Thus I need to detune CD3. In this test, I'll just

detune the reverse primer. Then I'll detune the forward primer later.

5

5x Buf527.5Two specimens used in this experiment

dNTP Q15.5Texas-22FS-06529.1229.9-0.78

enz mix15.5

Rox0.21.1

1867 5uM15.5

1869 10uM FQ15.5

RNaseP PPP*15.5

FS-2215.5

water6.837.4

total1899

*1852B

Address1870*WaterFHFH

1C35038.1360

2C44137.9560

3C53238.4860

4C62338.6560

5C71438.5560

MX4000 (BP)50(30')95(15'), 95(15")60(1') for 60 times

Wrong, I forgot to dilute 1870

Results:Since I added the wrong conc of 1870, this experiment is useless.

However, it is strange that Hex did not show up at all.

I believe I added RNaseP PPP.

Next:Repeat and do both 1867 and 1870.

080604-A(Bead temp)

072204-A(CD3 detune)

It may be useful to put CD3 mRNA and TREC DNA detection together for

a confirmatory test. Thus I need to detune CD3. In this test, I'll just

detune the reverse primer. Then I'll detune the forward primer later.

10

5x Buf560One specimen is used in this experiment

dNTP Q112Texas-22FS-06529.1229.9-0.78

enz mix112

Rox0.22.4

1869 10uM FQ112A=77 ul + 5.5 ul 1870 (5uM, 1:2 diluted from 10uM)

FS-22112B=77 ul + 5.5 ul 1867 (5uM)

water4.857.6

total14168Dilution of primers

1867 (5uM) 5ul + 45 ul water =0.5uM

1870 (10uM) 2.5ul + 47.5ul water = 0.5uM

MasterWater1867(0.5uM)1870 (0.5uM)StrengthFCtF

1010135.0528635

215ul A550.536.8425229

3730.338.8223022

4820.242.6121501

5910.149.0117290

6010134.5630710

715ul B550.534.626095

8730.335.3625282

9820.241.3518358

10910.156.745058

MX3000P (3270)50(30)95(15) then 95(15") 60(30") 60x

Results:1867 concentration needs to increase a little (to 200nM); 1870 (reverse primer)

can be at 100nM. The current format is 1867 at 100nM and 1870 at 200nM.

Just the reverse.

Thus in the confirmatory assay for the SCID identification, I can put CD3

and TREC together.

So far both TREC and CD3 probes are labeled with Fam and thus I could not

put them together. But a CY5 probe for CD3 has been ordered yesterday.

1877CD3-z ex5submitted 7/22/04CY5BHQ1agacgtggccgggaccct

Next:I'll have this CD3/TREC/RNaseP assay setup in two weeks and the KSU

student can use it for 200 tests.

nMForward (1867)Reverse (1870)

20035.0534.56

10036.8434.6

6038.8235.36

4042.6141.35

2049.0156.74

ForwardL use 80 nM

Reverse: use 80 nM

080604-A(Bead temp)

00

00

00

00

00

Forward (1867)

Reverse (1870)

080604-B(Q sol)

072204-B(Vogt)

072204-BCalib 1 1:100FS-106

072204-BCalib 2 1:300FS-107

072204-BCalib 3 1:900FS-108

072204-BCalib 4 1:2700FS-109

072204-BCalib 5 1:8100FS-110

072204-BCalib 6 1:24300FS-111

072204-BCalib 7 1:75000FS-112

072204-BTexas newborn 8FS-113



MX3000P (NK)

Ct TREC expected

Calib 1 1:100FS-10630.1528.2720274329030

Calib 2 1:300FS-10731.7728.3420917328531.7

Calib 3 1:900FS-10833.128.1421403336233.4

Calib 4 1:2700FS-10935.0228.0517613293735.1

Calib 5 1:8100FS-11036.6227.6917178345236.8

Calib 6 1:24300FS-11138.4527.5815294335038.5

Calib 7 1:75000FS-11242.0227.21175983596

Texas newborn 8FS-11336.7926.3266053175

Texas newborn 9FS-11437.4226.94280053069

Texas newborn 10FS-11537.1726.46277312888

(+) control water24.7360267980

606000

.

Dilutions

130.1528.271.88

331.7728.343.43

933.128.144.96

2735.0228.056.97

8136.6227.698.93

24338.4527.5810.87

72942.0227.2114.81

080604-B(Q sol)

0

0

0

0

0

0

0

080604-C(BeadsWash)

0

0

0

0

0

0

0

080604-D(Q sol)

072204-C(Hep2 CD3)

4

5x Buf522

dNTP Q14.4

enz mix14.4

18670.

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