brief introduction of nc22 plex str detection kit...2019/12/06 · Ø 4.3.3 humic acid • 10, 30,...

Brief Introduction of NC22 Plex STR Detection Kit

1. Five-color fluorescence detection platform, which can carry out multiple amplification and detection on 20 supplemental loci, 1 CODIS locus and 1 sex locus specified in the Standard of Judicial Identification;

2. Super high resolution, sensitivity and anti-inhibitor ability;

3. Rapid amplification, the amplification time is less than 1.5 hours.

4. Combined with our 21 Plex STR STR Detection kit, the number of detected loci can reach 40;

5. Meet the "Code for the Implementation of the Identification of Full Biological Syndrome" published by the Judicial Administration of Justice of the Ministry of Justice of PRC in 2014, it recommends to increase the number of detected loci from 19 to 29 or 39;

6.

7. Can be used in forensic analysis, kinship detection and scientific research for human genetic identification.


1.1 Features of NC22 Plex


1.2 Loci Arrangement Diagram of NC22 Plex

1.3 Analysis Software Loci Information Table of NC22 Plex GeneMapper ID-X


1.4 Electrophoretic Diagram of NC22 Plex Allelic LadderNC22 Plex Allelic Ladder contains 261 allelic genes


Component

PCR Amplification Kit

2.5× PCR Mix buffer containing anti-inhibition components such as MgCl2

NC22 Plex Primers primer mixtures for amplifying 22 loci

PCR Grade Water deionized ultra pure water

Control DNA 9947A(0.5ng/µL) standard positive control

Taq DNA Polymerase hot-start enzyme

PCR Detection KitNC22 Plex Allelic Ladder standard reference material for allele

genotyping

Orange-500 molecular weight internal labels with fluorescent markers

Matrix Kit 5 Dye Matrix used for fluorescence correction

Components of NC22 Plex STR Detection Kit

2.1 NC22 Plex STR Fluorescence Detection Kit (25µL×100 Reactions/Kit)

Component PCR Amplification Kit

2.5× PCR Mix 1000× 1

NC22 Plex Primers 500× 1

PCR Grade Water 1000× 1

Control DNA 9947A(0.5ng/µL) 20× 1

Taq DNA Polymerase 100× 1


Component PCR Detection Kit

NC22 Plex Allelic Ladder 20× 1

Orange-500 120× 1

Component Matrix Kit

5 Dye Matrix 50× 1

2.2 Components and Contents of NC22 Plex STR Detection Kit

95℃

3min94℃

15s60℃

1min

60℃

30min

4℃

∞30 Cycles

Component 25µL System（µL）

2.5× PCR Mix 10.0

NC22 Plex Primers 5.0

PCR Grade Water 8.0

Control DNA 9947(0.5ng/µL) 1.0

Taq DNA Polymerase 1.0

Total Volume 25.0


2.3 Reaction System and Procedures of NC22 Plex STR STR Detection Kit

Amplification Results of NC22 Plex STR Detection Kit

3.1 Amplification Results of Positive Control 9947A on 3130xl


3.2 Amplification Results of Blood on FTA on 3130xl


3.3 Amplification Results of Blood on Filter Paper on 3130xl


3.4 Amplification Results of Saliva on FTA on 3130xl

4.1 Sensitivity Test

Performance Measurement of NC22 Plex STR Detection Kit

Ø 4.1.1 Content of Extracted DNA • 1ng, 500pg, 250pg, 125pg, 62.5pg, 31.25pg • Repeat the experiment 3 times for all samples

Ø 4.1.2 Number of Direct Amplification Samples FTA Blood Card • 1, 2 or 3 slices - 1.2mm aperture • Repeat the experiment 3 times for all samples FTA Saliva Card • 1, 2 or 3 slices - 1.2mm aperture • Repeat the experiment 3 times for all samples


4.1.1 Extracted DNA

• 30 cycles• 25μL amplification system• 3130xl (3kV,10sec sampling time)• Baseline threshold 50RFU

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1ng 500pg 250pg 125pg 62.5pg 31.25pg

perc

ent o

f loc

i cal

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DNA Amount


4.1.1 Extracted DNA


4.1.2 Number of Direct Amplification Samples FTA Blood Card & FTA Saliva Card

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1 slice 2 slices 3 slices

perc

ent o

f loc

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Number of 1.2mm punches

blood on FTAbuccal on FTA



4.1.2 Number of Direct Amplification Samples FTA Blood Card & FTA Saliva Card

Ø 4.2.1 Mix Concentration • 0.75 x, 1 x, 1.25 x

Ø 4.2.2 Primer Concentration • 0.75 x, 1 x, 1.25 x

Ø 4.2.3 Annealing Temperature • 55℃, 57 ℃, 59 ℃, 61 ℃

Ø 4.2.4 Cycles Number • 28, 29, 30, 31

Ø 4.2.5 Reaction Volume • 25µl and 10µl


4.2 Tolerance Test

4.2.1 2 Concentrations of Mix and Primer



0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0.75X 1X 1.25X 0.75X 1X 1.25X

Perc

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f Loc

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Mix Concentration


4.2.1 Mix Concentration


4.2.2 Primer Concentration



4.2.3 Annealing Temperature


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

56℃ 58℃ 60℃ 61℃

Perc

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Annealing Temperature


4.2.3 Annealing Temperature — FTA Blood Card


4.2.3 Annealing Temperature — FTA Saliva Card


4.2.4 Cycles Number — 25 μL


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

28 29 30 31

Perc

ent o

f Loc

i Cal

led

Cycles Numbers (25uL)

blood on FTAbuccal on FTA提取DNA

4.2.4 Cycles Number — 10 μL


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

28 29 30 31

Perc

ent o

f Loc

i Cal

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Cycles Numbers (10uL)

blood on FTAbuccal on FTA提取DNA

4.2.4 Reaction Volume


Three kinds of inhibitors:

Ø 4.3.1 Heme • 0.1, 0.2, 0.3 and 0.4mM

Ø 4.3.2 Hemoglobin • 80, 100, 200 and 300µM

Ø 4.3.3 Humic Acid • 10, 30, 50 and 100ng/µL

4.3 Inhibition Test


• 30 cycles • 25μL amplification system• 3130xl (3kV,10sec sampling time)• Baseline threshold 50RFU

4.3 Inhibition Test


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0.1mM

0.2mM

0.3mM

0.4mM

60μM

100μ

M20

0μM

300μ

M

10ng

/μL

30ng

/μL

50ng

/μL

100n

g/μL

perc

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Hemoglobin

4.3.1 Heme


4.3.2 Hemoglobin


4.3.3 Humic Acid


1. Fast: Optimized components of PCR Reaction System that can shorten the PCR amplification time to less than 1.5 hours.

2. High resolution: Single tube reaction can get the correct typing of 22 loci at one time, which performs better than the other similar products in the market.

3. DNA extraction-free technology: Samples can be used for PCR amplification directly without extraction.

4. Higher rate of individual recognition and non-parent exclusion, showing excellent performance in criminal investigation, judicial identification and paternity test no matter NC22 Plex is used exclusively or jointly with other kits.

5. The product is fully domestically made, which improves both the independent innovation ability of the core technology of China's DNA detection reagents and the security of national DNA detection industry.

4. Summary

Summary of NC22 Plex STR Detection Kit

Comparison of Autosomal Supplementary Kits between Different Manufacturers

1. Loci Selected by Different Manufacturers

Comparison of Autosomal Supplementary Kits between Different Manufacturers

2. Comparison of Selected Loci

brief introduction of nc22 plex str detection kit...2019/12/06 · Ø 4.3.3 humic acid • 10, 30,...

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