pcr lcr elisa

8/8/2019 PCR LCR ELISA

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The gene probes are the extremely usefultools, probe signals are weak

This difficulty was overcome by PCR tool

It amplifies the DNA to 106 fold

Discovered by Kerry Mullis Its an enzymatic reaction used to generate

copies of target sequence

A series of temperatures have to be

maintained according to the DNA Exponential increase in the DNA

Visualized through various methods Efficiency of amplification is not perfect


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Gel Electrophoresis

SBYR greeny At the beginning the dye molecule weakly

fluorescene.y These molecules have strong affinity to the double

stranded molecules

y They bind to the dsDNA molecule and emit light

FRET: Fuorescence Resonance EnergyTransfery Two sequence specific oligonucleotides labeled with

fluorescent dyes

y Reporter dye and quencher

Detection by oligomer hybridisationy 32P- labled probe hybridizes in solution to one strand

of the denatured amplified product

y The probe target duplex is seperated from theunhybridized probe by polyacrylamide gel

electrophoresis and visualized by autoradiography


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1. Reverse Transcriptase PCR

2. Integrated Cell Culture PCR

3. Seminested, Nested and

Multiplex PCR4. Fingerprinting PCR

5. Real Time PCR

6. Amplified Fragment

Length Polymorphism PCR

7. Allele Specific PCR

8. Assembly PCR

9. Asymmetric PCR

10. Colony PCR11. Helicase Dependent

Amplification HDA

12. Hot Start PCR

13. Inverse PCR

14. In Situ PCR

15. ISSR PCR

16. Single Cell PCR

17. Touchdown PCR

18. Solid Phase PCR19. Quantative PCR


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Heat causes DNA strands to separateHeat causes DNA strands to separate

3

5

5

3Denature DNA strands 94Denature DNA strands 94ooCC

5 3

3 5


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Primers bind to the template sequence

Primers bind to the template sequence

TaqTaq Polymerase recognizes doublePolymerase recognizes double--stranded substratestranded substrate

3

55

3

Primers anneal 64Primers anneal 64ooCC

3

5

5

33 535


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Extend 72Extend 72ooCC

3

53

5

35

5

3

TaqTaq Polymerase extends primerPolymerase extends primer

DNA is replicatedDNA is replicated

RepeatRepeat denaturing, annealing, and extendingdenaturing, annealing, and extending 30 cycles30 cycles

3

53 535

5

3


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The Target Product Is Made In The

Third Cycle

3

55

3

5

3

53

Cycle 2

Cycle 33

3

3

35

5

5

5

Cycle 1

3

53535

5

3


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Mycobacterium tuberculosis

Putative insertion sequence designated asIS6110

Its present in high copy numbery

Its specific and has high repetitive nature makes it idealtarget for amplification by PCR

y The internal control DNA has been constructed whichuses same primer as target sequence

y Use of internal control DNA allows assessment of theefficacy of each individual reaction ensures that thereaction is not inhibited by interfering substances

y The PCR of internal control DNA is 600bp and easilydetectable from the 123kb of the product

y DNA fingerprinting ofM tuberculosis strain has beendeveloped using probes of IS6110


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Procedure

Sample Requirementsy Sputum, bronchoalveolar lavage specimen, and

bronchial washings.

The cells are lysed DNA extracted

PCR is performed

Amplification Concentrated, separate and electrophoresis

Analysis of PCR product


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Result

Load the sample with bromophenol blue

indicator and stain it with ethidium

bromide on the 12% polyacrylamide gel

Electrophoreses at 200V

Observe under UV transilluminator

y Positive: 123 bp fragment detect

y Negative: 600 bp fragment detected


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Target

IR

3

5

IR

Bam HIPvu II

Sal I

123bp

759-778

PCR PROCUCT

75881-862

T4


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Human Immunodeficiency Virus Type I

One of the two etiologic agent

PCR is highly sensitive

Discordance in the primer pairs include

y Sequence heterogenicityy Differential analytical sensitivity of the different

primes

y Short primers capable of accommodating

mismatchesy Sampling bias

y False positive result


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Guidelines

1. Highly conserved regions to be selected andamplified

2. Primers should be relatively long

3. To accommodate mismatches should have

3terminal

4. Annealing temperature which will provide

maximum mismatch stability

5. dUTP and uracil-N-glycosylase should be

incorporated into the amplification reaction to

minimize false positive PCR. This also helps to

increase specificity in amplification


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Procedure

Collection of blood sample

Processing

RT-PCR performed

Amplification

Concentrated, separate and electrophoresis

Detection by oligomer hybridisation


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HIV-1 9.7kb

HIV-1 gag791* 2299

SK462 SK431

SK102

(1366-1395)

123bp EXPECTED PCR PROCUCT142 bp

1366 1507

SK462 SK431SK102

(1403-1435) (1507-1481)


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Cryptococcus neoformans

Its region of the ribosomal DNA (rDNA) of severalisolates ofCneoformans

Three specific primers of the fungi Cneoformans

The primers do not amplify the sequence fromother sequences

They have the potential to recognize the fungi inthe clinical specimens

PCR fingerprinting: one primer is used todistinguish between the two varieties of the fungi

This procedure allows the isolates to befingerprinted


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Type Name Nucleotice sequence , 5 TO 3 Location on target

C. Neoformans

identification

NS1 GTAGTC ATA TGC TTG TCT C 5

NA8 TTC GCA GGT TCA CCT ACGGA 3

General ITS1 TTC GTAGGT GAA CCT GCG A 5

ITS4 TCC TCC GCT TAT TGA TAT GC 3

C. Neoformans specific CN4 ATC ACC TTC CCA CTA ACA CAT T 3

CN5 GAAGGG CAT GCC TGT TTG AGAG 5

CN6 TTT AAGGCG AGC CGA CGT CCT T 3

PCR fingerprinting (GTG) Random

(GACA) Random

GAGGGT GGC GGT TCT Random


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Procedure

DNA extraction

PCR

Preperation of primersAmplification

Concentrated, separate and electrophoresis

The result is given in the figure


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Giardia Lamblia

Its is the only species being transmitted

to the succesive genration

It has a unique 18s rRNA which is one of

the most ancient

It contains many species specific

regions

Assay detects the 18s rRNA genesequence in fecal specimens obtained


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Function Name Nucleotice sequence , 5 TO 3 Location

Upstream primer JW1 GCG CAC CAGGAA TGT CTTGT 1251-1270

JW2 TCA CCT ACGGAT ACC TTG TT 1433-1414

Internal probe RDR34 AGGGAC GCG TCC GGC G 1306-1291

Primer and probe sequence for the

detection of the parasite


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Procedure

The fecal specimens collected

Sample preperation

Extraction of the DNA PCR amplification

Detection and analysis of the amplified

product


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1251-1270

JW1

1251-1270

JW2

13906-1291

Probe RDR34

Internal oligonucleotice

probe

G. Lamblia 18S rRNA gene 35

EXPECTED PCR PROCUCT183bp

1251 1433


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Advantages Disadvantages

Detect specific nucleic

acid

Sensitive, large target Independent access to

viable and nonviable

Optimises

Fingerprinting allows todiscriminate the products

Access level of gene

expression

Inhibition due to humic

substances and metals

Non specific priming Infectious and

noninfectious mixture of

the sample


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Introduction

Whiteley et al. # Wu and Wallace

Target dependent ligation

LCR employs a thermostable DNA polymeraseand thermostable DNA ligase

It uses four oligonucleotide probes of 20-24b each

Each pair of the oligonucleotide is designed tobind to the denatured target DNA

A pair of oligonucleotides are made to bind to oneof the DNA target strands

A second pair of oligonucleotides is designed tohybridize to the same regions on the

complementary DNA.


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The oligonucleotides are mixed with extractedtarget DNA, denatured

The reaction is then cooled allowing theolingnucleotide probes to bind to the target DNA

DNA polymerase fills the gap and linked by DNAligase yielding doublestranded DNA molecule

40-50bp length The cycle is repeated 30 -40 times

The power of LCR is its compatibility with otherreplication-based amplification methods.

By combining LCR with a primary amplification,one effectively lines up the crosshairs todistinguish single base-pair changes withpinpoint accuracy.


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A

T

G

A

T

C

T

A

AT

G

G

AT

GC

Denature the DNA 94C

Anneal the oligonucleotides 65C

Ligate with thermostable DNA ligase 65C

A

T

G

C


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T

A

T

A

Repeat the cycle 20-30 times

C

AT

TA

G

DNA is denatured at 94~ and the four LCRprimers anneal to their complementary strands at 65~ which is approximately 5~

below theirTin. Thermostable ligase (Q) will only ligate primers that are perfectly complementary to their target sequence and

hybridize directly adjacent to each other (as shown withL. monocytogenes, left). The discriminating bases at the 3 ends of the

upstream primers are depicted as boxes on the target as well as on the primers for clarity. Primers that have at least a single

base-pair mismatch at the 3 end contributing to the junction of the two primers will not ligate (as shown with L. innocua, right).

The discriminating primers have a 2-bp noncomplementary AA tail at their 5ends to avoid ligation of the 3 ends.


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Principle of LCR. (Bottom) The example shown is an LCR with matched target(L.monocytogenes) and

mismatched target(L. innocua). The pathogenic bacteria L. monocytogenes canbe distinguished from other

closely relatedListeria spp. (e.g., L. innocua) by a single base-pairdifference in the 16S rDNA/2~L.

monocytogenes has an A-T base pair at nucleotide 1258, whereas L. innocua has a G-C base pair at this

position.


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N gonorrhoeae

The assay amplification kit uses four

probes complementary to a 48-bp region

of the gonococcal opacity 1 (opa-1)

gene

(GenBankaccession name, NGOOPC

B) This gene sequence is repeated up to

11 times in the N. gonorrhoeae genome. The chosen sequence is specific for

Ngonorrhoeae.


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To resolve discrepant culture-negative,

LCR-positive specimens, another LCR assay with the pilin probe set specific

forN. gonorrhoeae was used.

The pilin probe set is specific for N. gonorrhoeae, as described previously

(1a). The specificity of the pilin LCR assay forN. gonorrhoeae was greater

than 99% when it was further evaluated with clinicalspecimens.

The pilin LCR assay procedure was similar to theN. gonorrhoeae LCR assay

procedure as described above, except that thermocyclingwas for 53 cycles of

858C for 30 s and 608C for 1 min and 100 ml of each sample diluted 1:1 with

N gonorrhoeae LCR assay transport buffer was assayed.

Specimens that gave duplicate IMx signals above 100 counts per second per

second were confirmed asN. gonorrhoeae LCR assay positive. To resolve the

results for specimens that were culture positive.

N gonorrhoeae LCR assay negative, samples were diluted with specimentransport buffer and were retested by theN. gonorrhoeae LCR assay. The

bacterial isolates, when available, were also heated in specimen transport

buffer and were tested by theN. gonorrhoeae LCR assay.


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Important factors

Design of Primers

y Single basepair overhangs

y Temperature range

y Unique primers

y Noncomplementary tails

Conditions

y One set of four primersy Thermostable ligase

y There is no extention


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Detection methods for product

y Labeled with biotin 5end and nonisotopic

reporter at 3 endy Dye include fluorescein dye in blue (FAM, 5-

carboxyfluorescein) and digoxigenin.

y anti-digoxigenin antibodies coupled to alkaline

phosphatase (AP) greatly improved thesensitivity.

y Subsequent detection of the AP could be

achieved using colorimetric, fluorescent, or

luminogenic substrates.

Applications

y Genetic , viruses and bacterial diseases


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Limitations

Background amplification due to blunt

end ligation of probe duplexes

Long reactions

Carryover contaminations

Nonstringent annealing conditions


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Types of ELISA

1. Direct ELISA

2. Indirect ELISA

3.Sandwich ELISA

4. All three systems can be used to form

the basis of a group of assays called

competition or inhibition ELISAs.

5. Reverse ELISA

6. ELISA spot


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Direct ElISA

Antigen added to solid phase, incubation

Non bound antigens washed

Antigen specific antibody with enzymeconjugate added which binds antigen

unbound antibody washed

Substrate/ chromophore added

Enzyme catalyzes reaction to give colour


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Indirect ELISA

Antigen added to solid phase, incubation

Non bound antigens washed

Antigen specific antibody added which

binds antigen

Antibodies labeled with enzyme

(conjugate) which are antispecies

Excess is washed off Substrate/ chromophore added

Enzyme catalyzes reaction to give colour


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Sandwich ELISA

Direct sandwich Indirect sandwich

Antibodies, Wash

Antigen sp, wash

Antibody enzyme

conjugaete

Substrate

Enzyme catalyzed to

give colour

Antibody, wash

Antigen sp, wash

Antibody sp to antigen

Anti-antibody enzyme

conjugate

Substrate

Enzyme catalyzed to

give colour


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Competitive ELISA Incubate antibody with antigen

Add antigen-antibody mixture to the well coated withantigen (The more antigen in the sample, the lessantibody will be able to bind to the antigen in the well,hence "competition)

Wash

Add enzyme conjugate secondary antibody

Wash

Add substrate

Enzyme catalyzes the reaction to give colour

For competitive ELISA, the higher the original antigenconcentration, the weaker the eventual signal.

The major advantage of a competitive ELISA is theability to use crude or impure samples and stillselectively bind any antigen that may be present.


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Enzymes : Chromogens /Substrates

Horse radish peroxidase

Ortho phenylene diamine/ hydrogen peroxide

2,2azino-diethylbenzaldehyde/hydrogen peroxide

Tetra methyl benzidine/ hydrogen peroxide

5-amino salicylic acid / hydrogen peroxideDiaminobenzidine/ hydrogen peroxide

Alkaline phosphatase

Para-nitro phenylphosphate

-GalactosidaseOrtho-nitrophenyl -galactosidase

Urease

Bromocresol purple


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Influenza virus H1N1

Antigen coated microtitre plate

Diluted control serum and test sample are added

If the sample contains antibodies IgG it will bindto the microtitre plate

Unbound are washed out

Enzyme conjugate: Anti human IgG peroxidaseconjugate added

Unbound antibodies washed Substrate :Tetramethylbenzidine is added

Blue coloured soluble product which turns intoyellow after adding the acid stopping solution


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M tuberculosis

ELISA methodology for measurement of

IgGAb are available.

38-Kd Ag provides serodiagnostic test

with most favorable test characteristics

described, but is limited by the lack of

purified Ag.

Serum IgGAb are observed to riseduring the first 3 months of therapy but

fall after 12-16 months.


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Reverse ELISA


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DisadvantagesAdvantages

pcr lcr elisa

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