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Method for detecting nucleic acid mass of sample

A detection method and sample technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of indetermination of influence degree, inability to detect DNA degradation, etc., and achieve the effect of expanding the range of reagents

Active Publication Date: 2011-07-27
SUZHOU NUHIGH BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the difference in the length of the DNA template, the above three kits cannot detect the degradation of DNA in the sample, and DNA degradation is one of the important parameters for DNA quality assessment, and it is impossible to determine the influence of inhibitors in the sample on the subsequent forensic DNA detection.

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  • Method for detecting nucleic acid mass of sample
  • Method for detecting nucleic acid mass of sample
  • Method for detecting nucleic acid mass of sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: Evaluate the degradation situation of biological sample with the method of the present invention

[0051] Experimental scheme: In a 25ul fluorescent quantitative PCR reaction system, there are 2 primers for amplifying the RPPH1 gene (the nucleotide sequences of which are shown in SEQ ID No.1 and SEQ ID No.2 respectively) and 1 FAM-labeled probe (The nucleotide sequence is respectively shown in SEQ ID No.3);

[0052] 2 primers for amplifying the Fas gene (its nucleotide sequence is shown in SEQ ID No.4 and SEQID No.5 respectively) and 1 HEX-labeled probe (its nucleotide sequence is shown in SEQ ID No.6 respectively) shown);

[0053] 2 primers for amplifying internal control IPC1 (its nucleotide sequences are shown in SEQ ID No.7 and SEQ ID No.8 respectively) and 1 TAMRA-labeled probe (its nucleotide sequences are shown in SEQ ID No .9);

[0054] 2 primers for amplifying internal control IPC2 (its nucleotide sequences are shown in SEQ ID No.10 and SEQ ID ...

Embodiment 2

[0063] Embodiment 2: Evaluate biological samples affected by inhibitors with the method of the present invention

[0064] Referring to Example 1, to simulate actual samples, the PCR inhibitor humic acid (Humic acid), which is widely present in soil, plants and other environments, is added to the fluorescent quantitative PCR reaction system of Example 1, and all systems contain the same human The amount of genomic DNA, and the obtained Ct value of the reaction reflects the extent to which the PCR system is subjected to inhibitors. The data are shown in Table 2:

[0065] Table 2, method evaluation sample of the present invention is influenced by inhibitor

[0066]

[0067] The data in Table 2 shows that in PCR, the same inhibitor concentration has different degrees of inhibition on IPC fragments of different lengths, and the inhibition degree of long fragments is greater than that of small fragments. For example, in the presence of 3ng / ul humic acid, the Ct value of IPC1 is...

Embodiment 3

[0069] Embodiment 3: Evaluate the degradation situation of biological sample with the method of the present invention

[0070] Select 2 DNA fragments (non-overlapping regions) in the human gene TH01 (intron1 of human tyrosine hydroxylasegene), the nucleotide sequences of which are shown in SEQ ID No.23 and SEQ ID No.24 respectively, and with reference to Example 1, perform fluorescence Quantitative PCR detection. Amplification reagent components include:

[0071] (1) 2 primers for amplifying TH01 gene (its nucleotide sequence is shown in SEQ ID No.13 and SEQ ID No.14 respectively) and 1 FAM-labeled probe (its nucleotide sequence is as shown in SEQ ID No.14) IDNo.15), the product is 135bp;

[0072] (2) 2 primers for amplifying TH01 gene (its nucleotide sequence is shown in SEQ ID No.16 and SEQ ID No.17) and 1 HEX-labeled probe (its nucleotide sequence is as shown in SEQ ID No.17) IDNo.18), the product is 313bp;

[0073] (3) 2 primers for amplifying the internal control IPC1...

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Abstract

The invention relates to the technical field of biology, and discloses a method for detecting the nucleic acid mass of a sample, which comprises the following steps: adding internal control nucleic acid molecules with different lengths into a sample; carrying out specific amplification on conserved sequences with different lengths of nucleic acids to be detected in the sample and the internal control nucleic acid molecules in the same reaction system; detecting the amplification results; comparing the quantity of the conserved sequences and the quantity of the internal control nucleic acid molecules; and analyzing. Preferably, the invention adopts the real-time fluorescent quantitative PCR (Polymerase Chain Reaction) technology in the amplification and detection steps. The invention also provides a kit for detecting the nucleic acid mass. By detecting DNA (deoxyribonucleic acid) segments with different lengths to determine the DNA degradation degree in the sample, the method disclosedby the invention can accurately reflect the incidence of the sample inhibitor, and can be widely used in the fields of medicolegal examination, microbiological examination and food safety.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting the quality of nucleic acid in a sample. Background technique [0002] DNA (Deoxyribonucleic acid, deoxyribonucleic acid) is a substance widely used in forensic testing. DNA carries the genetic information of humans or other species and is present in various samples such as blood, tissue, secretions, etc. In forensic DNA testing, available biological samples obtained from the scene are rarely "clean" and contain impurities such as bacteria and chemicals that may affect DNA testing. In addition, DNA testing of low copy number DNA and / or highly degraded DNA samples is also one of the difficult problems in forensic DNA testing. Based on the above two problems, it is necessary to evaluate the quality of biological samples before conducting forensic DNA testing. The evaluation includes: (1) the copy number and degradation degree of DNA in the sample; (2) whether t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 毕万里卢红波
Owner SUZHOU NUHIGH BIOTECH
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