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Emb B gene mutation detection specific primers and liquid phase chip

A detection solution and specific technology, applied in the field of molecular biology, can solve the problems of poor stability and repeatability, high false positive rate, high price, etc., and achieve the effects of simple steps, avoiding cross-reaction, and improving accuracy.

Inactive Publication Date: 2011-04-13
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the detection methods for emb B gene mutation mainly include polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), PCR restriction fragment length polymorphism (PCR-RFLP), PCR direct sequencing, etc. There are disadvantages such as low sensitivity, high false positive rate, poor stability and repeatability, and high price, which cannot meet the needs of practical applications

Method used

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  • Emb B gene mutation detection specific primers and liquid phase chip
  • Emb B gene mutation detection specific primers and liquid phase chip
  • Emb B gene mutation detection specific primers and liquid phase chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1e

[0019] Embodiment 1 emb B gene mutation detection liquid chip mainly includes:

[0020] 1. ASPE Primers

[0021] Mutation site for emb B gene: Phe(TTC)→Leu(TTA) of codon 285, Met(ATG)→Ile(ATC / ATA / ATT) or Leu(CTG) or Val(GTG) of codon 306, codon Phe(TTC)→Val(GTC) of 330, Gly(GGC)→Ala(GCC) or Asp(GAC) or Cys(TGC) or Ser(TCC) of codon406, Gln(CAG)→Arg(CGG) of codon 497 ), respectively design specific primer sequences. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0022] Table 1 ASPE primer sequence (Tag sequence + specific primer sequence)

[0023]

[0024]

[0025] 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...

Embodiment 2e

[0036] Example 2 emb B gene mutation detection liquid chip detection of samples

[0037] The formula of described various solutions is as follows:

[0038] 50mM MES buffer (pH5.0) formulation (250mL):

[0039] Reagent

source

Final concentration

Dosage per 250mL

MES(2[N-Morpholino]

Sigma M-2933

0.05M

2.44g

[0040] ethanesulfonic acid)

5MNaOH

Fisher SS256-500

---

5 drops

[0041] 2×Tm hybridization buffer:

[0042] Reagent

source

Final concentration

Dosage per 250mL

1M Tris-HCl, pH8.0

SigmaT3038

0.2M

50mL

[0043] 5MNaCl

Sigma S5150

0.4M

20mL

Triton X-100

Sigma T8787

0.16％

0.4mL

[0044] Store at 4°C after filtration.

[0045] ExoSAP-IT kit was purchased from US USB Company.

[0046] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service C...

Embodiment 3

[0111] The liquid phase chip of embodiment 3 different ASPE primers is to the detection of emb B gene mutation site

[0112] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0113] Taking the Phe(TTC)→Leu(TTA) of codon 285 of emb B gene and the Phe(TTC)→Val(GTC) mutation detection liquid chip of codon 330 as an example, the wild type and mutation of codon 285 and 330 were respectively targeted The specific primer sequence at the 3' end of the ASPE primer is designed, and the Tag sequence at the 5' end of the ASPE primer is selected from SEQ NO.1-SEQ NO.17. Correspondingly, the complementary paired tag sequence coated on the microsphere The anti-tag sequence is selected from SEQ NO.35-SEQ NO.51. The specific design is shown in the following table (Table 8). 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.

[0114...

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Abstract

The invention discloses ethambutol (emb) B gene mutation detection specific primers and a liquid phase chip. The liquid phase chip comprises ASPE primers, microspheres and amplification primers, wherein the specific primers of the ASPE primers are selected from SEQ ID No.18 and SEQ ID No.19 at the codon 285th site, SEQ ID No.20 and more than one of sequences from SEQ ID No.21-25 at the codon 306th site, SEQ ID No.26 and SEQ ID No.27 at the codon 330th site, SEQ ID No.28 and more than one of sequences from SEQ ID No.29-32 at the codon 406th site, and / or SEQ ID No.33 and SEQ ID No.34 at the codon 497th site. The detection result of the emb B gene mutation detection liquid phase chip provided by the invention is completely matched with that of a sequencing method, and the liquid phase chip has good signal-noise ratio.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a specific primer for emb B gene mutation detection and a liquid phase chip. Background technique [0002] Ethambutol (EMB), a synthetic drug discovered in 1961 with antimycobacterial activity, is the first-line anti-tuberculosis drug. EMB is an arabinose analog, which acts on the target molecule arabinosyltransferase, inhibits the polymerization of arabinose into arabinogalactan, thereby affecting the complex of mycolic acid-arabinogalactan-peptidoglycan in the cell wall The formation of substances leads to the death of bacterial cells, and it also makes it easier for drugs (such as rifampin) with target molecules in the cells to enter the cells, and has a synergistic anti-tuberculosis effect when used in combination with rifampicin. Recent studies have shown that tuberculosis resistance to EMB is related to the mutation of the embABC oper...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森罗彩英甘丹翠陈家欣
Owner SUREXAM BIO TECH
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