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Specific primer and liquid phase chip for rpoB gene mutation detection

A detection solution and specificity technology, applied in the field of molecular biology, can solve the problems of uncertain mutation position, high false positive rate, easy contamination of samples, etc., achieve good signal-to-noise ratio, avoid cross-reaction, and have consistent detection results Effect

Inactive Publication Date: 2013-01-30
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, the detection products of rpoB gene mutation mainly include real-time fluorescent quantitative PCR, direct sequencing, denaturing gradient gel electrophoresis, DHPLC, etc., which have disadvantages such as low sensitivity, easy contamination of samples, high false positive rate, uncertainty of mutation position, and high price. , at the same time, due to the limitation of the detection flux, it cannot meet the needs of practical applications

Method used

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  • Specific primer and liquid phase chip for rpoB gene mutation detection
  • Specific primer and liquid phase chip for rpoB gene mutation detection
  • Specific primer and liquid phase chip for rpoB gene mutation detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The rpoB gene mutation detection liquid chip mainly includes:

[0022] 1. ASPE Primers

[0023] Eight common mutation sites for rpoB gene: codon513(CAA→AAA), codon 516(GAC→GTC), codon 522(TCG→TTG), codon 526(CAC→TAC / CTC / GAC / CGC), codon 529 (CGA→CCA), codon 531(TCG→TGG / TTG), codon 533(CTG→CCG), codon 545(CTG→ATG), respectively design specific primer sequences. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0024] Table 1 ASPE primer sequence (Tag sequence + specific primer sequence)

[0025]

[0026]

[0027]Each ASPE primer includes two parts, the 5' end is a specific tag sequence for the anti-tag sequence on the corresponding microsphere, and the 3' end is a mutant or wild-type specific primer fragment (as shown in the above table 1). All ASPE primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Each synthesized primer was prepared into a stock soluti...

Embodiment 2

[0039] Example 2 Detection of samples by rpoB gene mutation detection liquid chip

[0040] The formula of described various solutions is as follows:

[0041] 50mM MES buffer (pH5.0) formulation (250mL):

[0042]

[0043] 2×Tm hybridization buffer:

[0044]

[0045] Store at 4°C after filtration.

[0046] ExoSAP-IT kit was purchased from US USB Company.

[0047] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0048] 1. Sample DNA extraction:

[0049] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0050] 2. PCR amplification of samples to be tested

[0051] Two pairs of primers were designed using Primer5.0, and the size of the rpoB gene product was amplified by one-step multiplex PCR with a size of 392 bp. The primer sequences (SEQ ID NO.61-62) are shown in Table 3 above.

[0052] First prepare the multiplex PCR primer working solution: take 100 uL of th...

Embodiment 3

[0112] The detection of the rpoB gene mutation site by the liquid chip of embodiment 3 different ASPE primers

[0113] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0114] Taking the rpoB gene codon513(CAA→AAA), codon526(CAC→TAC), codon533(CTG→CCG) site mutation detection liquid chip as an example, ASPE primers were designed for the wild-type and mutant types of codon513, codon526 and codon533 respectively3 The specific primer sequence at the 'end, and the Tag sequence at the 5' end of the ASPE primer is selected from SEQ ID NO.1-SEQ ID NO.20, correspondingly, the anti-tag sequence coated on the microsphere is complementary to the corresponding tag sequence Selected from SEQ ID NO.41-SEQ ID NO.60. The specific design is shown in the following table (Table 9). The synthesis of ASPE primers, microspheres coated with anti-tag sequences, amplification primers, detection methods, etc. are as described in Example 1 and Example 2.

...

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Abstract

The invention discloses a specific primer and a liquid phase chip for RNA polymerase beta-subunit (rpoB) gene mutation detection. The liquid phase chip mainly comprises an ASPE primer consisting of a tag sequence of an end 5' and the specific primer of end 3' aiming at a mutant site, microspheres which are coated with specific anti-tag sequences and provided with different color codes respectively, and an amplification primer, wherein the specific primer is more than one of SEQ IDNO.21 and SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, SEQ ID NO.25 and SEQ ID NO.26, SEQ ID NO.27 and SEQ ID NO.28 to SEQ ID NO.31, is more than one of SEQ ID NO.32 and SEQ ID NO.33, SEQ ID NO.34 and SEQ ID NO.35 to SEQ ID NO.36, is SEQ ID NO.37 and SEQ IDNO.38, and / or is SEQ ID NO.39 and SEQ ID NO.40; and the tag sequence is a sequence selected from SEQ ID NO.1 to SEQ ID NO.20. The detection result of the liquid phase chip provided by the invention is completely consistent with that of a sequencing method. The prepared liquid phase chip for the rpoB gene mutation detection has high signal-noise ratio and can realize the parallel detection of a plurality of mutant sites.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a rpoB gene mutation detection specific primer and a liquid phase chip. Background technique [0002] The rpoB gene (RNA Polymerase β-Subunit Gene) is the gene encoding the β subunit of RNA polymerase, with a total length of 3543 bases. Among them, the molecular mechanism of Mycobacterium tuberculosis resistance to rifampicin is related to the rpoB gene, and rifampicin passes through Binds to the β subunit of RNA polymerase, thereby hindering the transcription and elongation of RNA. The core region of this gene encoding the 507-533 amino acid site is the rifampicin resistance-determining region (RRDR). When mutations occur in this region, including point mutations or short insertion and deletion mutations, DNA-dependent Rifampicin cannot bind to the β subunit of bacterial RNA polymerase, thus showing drug resistance. [0003] Mutations of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森甘丹翠邓晶晶刘志明
Owner SUREXAM BIO TECH
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