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PCR method using two groups of homologous primer and its reaction liquid and use

A primer set and chain reaction technology, applied in the field of molecular biology, can solve the problems that false negatives or non-transformed products cannot be avoided at the same time, and achieve the effect of eliminating false negatives and expanding the detection range

Inactive Publication Date: 2004-07-14
徐定邦 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The technical problem to be solved by the present invention is to provide a polymerase chain reaction method using two sets of homologous primers and its reaction solution and its application in the preparation of medical microbial nucleic acid detection reagents, so as to break through the prior art in the same The routine of only one pair of forward and reverse primers for the source gene overcomes the defects that false negatives or non-transgenic products cannot be avoided at the same time in the detection of homologous genes

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 takes the sequence of known HBV variants as an example, using two sets of homologous primer methods to overcome false negatives.

[0031] The designed forward primer set and reverse primer set include three primers. Table 1 is based on the HBV nucleic acid sequence (E00010, see nucleotide sequence list), and lists the length, position and other basic characteristics of each primer.

[0032] Table 2 lists the sequence of each primer. The bases with darkened font indicate bases that are not exactly the same as each other in a set of homologous primers.

[0033]

[0034] Name

[0035] The data in Table 1 and Table 2 show that the maximum difference in primer length between the positive primer set is 4 bases, and the length of the three primers in the reverse primer set is exactly the same. The sequence homology between primers in each set of primers exceeds 90%.

[0036] Table 3 analyzes the match between the positive and negative primer sets and...

Embodiment 2

[0047] The PCR method of two sets of homologous primers was used to test 110 HBV serum samples, and the negative control was 5 normal human serums (previously tested by ELISA for HBsAg, HBsAb, HBcAb, HBeAg and HBeAb). The PCR reaction uses Clontech's Advantage-2 kit. Take 200μL of serum from each sample and add an equal volume of lysis buffer (the formula is Tris.HCl, 8%, Tween20, 2%), mix well, and lyse in a water bath at 95°C for 15 minutes. Take it out, centrifuge at 12000 rpm for 15 min, and take the supernatant for PCR reaction. Reaction mother liquor: Taq enzyme 1μL, dNTP1μL, 10Xbuffer, 5μL, 2 groups of six primers in Table 2 each 2uL to make the concentration 0.5μM, dd water 3μL. Add 2μL of reaction mother solution and 3μL of serum lysate to each tube. Reaction conditions: first denature at 95°C for 2 minutes, then carry out 30 cycles at 95°C 30sec-72°C 30sec-72°C 30sec, and finally extend at 72°C for 2 minutes. The electrophoresis test results showed that all HBV sera obta...

Embodiment 3

[0049] Example 3 is to design a PCR method with two sets of homologous primers to determine the total lactobacilli in the human vagina. Vaginal microecology is a complex system containing many microbial populations. Among them, the micro pH environment and H 2 O 2 It plays an important role in the prevention and treatment of diseases including AIDS. There are dozens of species of Lactobacillus. Among the resident Lactobacilli that have been found in the human vagina are more than ten species such as L.acidophillus (see Table 9). At present, genes encoding 16sRNA are commonly used to identify microbial species. There are no fewer than tens of thousands of sequences in the gene bank. The 16sRNA gene contains about 1500 bases (see the nucleotide sequence list), and no primer can be found in any region of the gene, which can completely match the 16sRNA gene sequence of more than a dozen strains listed in Table 9 or It is highly matched and completely mismatched or seriously mismatched...

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Abstract

The present invention belongs to the field of molecular biology technology, and provides PCR process with two groups of homologous primers and its reaction liquid. In the PCR process, two groups, including one forward group and one reverse group, of homologous primers, designed based on the identical segment of known destination gene are used. Each of the groups includes 1-3 primers with 0-3 template mismatching bases and length difference of 0-10 nucleotides. In the genes, 5' starting positions differ by 0-10 nucleotides. Owing to designing the primer groups strictly based on the known tested sequence, the present invention can reduce or even eliminate the false negative in homologous gene PCR detection and thus has wide application foreground in preparing nucleic acid detecting reagent for medical microbes, such as virus, bacteria, etc.

Description

Technical field [0001] The invention relates to molecular biology technology, in particular to a polymerase chain reaction method and its reaction solution and its application in preparing medical microbial nucleic acid detection reagents. technical background [0002] The classic polymerase chain reaction method was invented in the mid-1980s. It uses one forward and one reverse primer to amplify a specific gene. For more than ten years, several PCR methods using two or more primers at the same time have been developed. Among them, there are mainly multiple primer PCR and degenerate primer PCR. [0003] Multiplex PCR is to simultaneously amplify two or more target genes in a reaction tube. The sequence of each pair of primers in multiplex PCR matches the corresponding target gene, and there is no homology between the primers. Multiplex PCR has also been used to amplify different regions of the same gene, but the primer pairs need to be far apart, otherwise they will interfere wit...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/689C12Q2600/16
Inventor 徐定邦朱德芬谢文凯徐文慧
Owner 徐定邦
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