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 problem of not being able to avoid false negatives or non-transformed products at the same time, and achieve the effect of eliminating false negatives and expanding the detection range.

Inactive Publication Date: 2007-08-29
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 primers to overcome false negatives.

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

[0032] Table 2 lists the sequence of each primer, and bases in bold font indicate bases that are not completely identical to each other in a group of homologous primers.

[0033] Table 1. Names and basic characteristics of six HBV primers

[0034]

[0035] Table 2. The sequence of six hepatitis B virus primers

[0036] name

[0037] The data in Table 1 and Table 2 show that the difference in the length of the primers in the forward primer group is at most 4 bases, and the lengths of the three primers in the reverse primer group are exactly the same. The sequence hom...

Embodiment 2

[0052] 110 HBV serum samples were detected by the PCR method with two sets of homologous primers, and the negative control was 5 normal human serum samples (HBsAg, HBsAb, HBcAb, HBeAg and HBeAb were all negative by ELISA in advance). The PCR reaction uses the Advantage-2 kit from Clontech Company. Take 200 μL of serum from each sample and add an equal volume of lysate (Tris.HCl, 8%, Tween20, 2%), mix well, and lyse in a water bath at 95° C. for 15 minutes. Take it out, centrifuge at 12000rpm for 15min, and take the supernatant for PCR reaction. Reaction master solution: 1 μL of Taq enzyme, 1 μL of dNTP, 5 μL of 10× buffer, 2 uL of each of the six primers in the 2 groups in Table 2 to make the concentration 0.5 μM, 3 μL of dd water. Add 2 μL reaction master solution and 3 μL serum lysate to each tube. Reaction conditions: denature at 95°C for 2 minutes, then perform 30 cycles at 95°C for 30sec-72°C for 30sec-72°C for 30sec, and finally extend at 72°C for 2min. Electrophoresi...

Embodiment 3

[0054] Embodiment 3 is designed to measure the total lactobacillus of human vagina with two groups of homologous primer PCR method. The vaginal microbiome is a complex system containing many microbial populations. Among them, the micropH environment caused by the activity of lactobacilli and the 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, and more than ten species of resident Lactobacillus have been found in the human vagina, including L. acidophilus (see Table 9). Currently, the gene encoding 16sRNA is commonly used to identify microbial species. There are no less than tens of thousands of sequences included in the gene bank. The 16sRNA gene contains about 1500 bases (see the nucleotide sequence list), no matter in which region of the gene, no primer can be found, which can completely match the 16sRNA gene sequence of more than a dozen bacterial species listed in Table 9 or Hi...

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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 the preparation of medical microbial nucleic acid detection reagents. technical background [0002] Invented in the mid-1980s, the classic polymerase chain reaction method uses a forward primer and a reverse primer to amplify a specific gene. Over the past decade, several PCR methods using two or more primers have been developed, among which multiple primer PCR and degenerate primer PCR are the main ones. [0003] Multiplex PCR is to simultaneously amplify two or more target genes in one 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 interfere with each othe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/00C12P19/34C12Q1/68
CPCC12Q1/689C12Q2600/16
Inventor 徐定邦朱德芬谢文凯徐文慧
Owner 徐定邦
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