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Specific primer and liquid-phase chip for katG, inhA and ndh genetic mutation detection

A detection solution and specific technology, applied in the field of molecular biology, can solve the problems of inability to determine the mutation position, easy contamination of samples, and high false positive rate, achieve good signal-to-noise ratio, consistent detection effect, and avoid cross-reaction effects.

Inactive Publication Date: 2013-03-20
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the detection products of katG, inhA and ndh gene mutations mainly include real-time fluorescent quantitative PCR, direct sequencing, denaturing gradient gel electrophoresis, DHPLC, etc., which have low sensitivity, easy sample contamination, high false positive rate, and inability to determine the mutation position. High price and other shortcomings, and due to the limitation of detection flux, it cannot meet the needs of practical applications

Method used

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  • Specific primer and liquid-phase chip for katG, inhA and ndh genetic mutation detection
  • Specific primer and liquid-phase chip for katG, inhA and ndh genetic mutation detection
  • Specific primer and liquid-phase chip for katG, inhA and ndh genetic mutation detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 The liquid chip for detecting mutations of katG, inhA and ndh genes mainly includes:

[0024] 1. ASPE Primers

[0025] For the wild type and mutant types of the mutation site codon 315AGC→ACC / ACA / AAC / ATC / CGC and G463T of the katG gene, the mutation site C(-15)T of the inhA gene, and the mutation site A110G and G268A of the ndh gene, respectively Design specific primer sequences. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0026] Table 1 ASPE primer sequence (Tag sequence + specific primer sequence)

[0027]

[0028]

[0029] 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 w...

Embodiment 2

[0042] Example 2 Detection of samples by the katG, inhA and ndh gene mutation detection liquid chip described in Example 1

[0043] The formula of described various solutions is as follows:

[0044] 50mM MES buffer (pH5.0) formulation (250mL):

[0045]

[0046] 2×Tm hybridization buffer:

[0047]

[0048] Store at 4°C after filtration.

[0049] ExoSAP-IT kit was purchased from US USB Company.

[0050] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0051] 1. Sample DNA extraction:

[0052] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0053] 2. PCR amplification of samples to be tested

[0054] Using Primer5.0 to design five pairs of primers, one-step multiplex PCR amplified the katG gene mutation site (AGC315ACC / ACA / AAC / ATC / CGC) and G463T, inhA gene mutation site C(-15)T and ndh gene Two mutation sites A110G and G268A, a total of five target sequences...

Embodiment 3

[0112] Example 3 Detection of katG, inhA and ndh gene mutation sites by liquid chip with different ASPE primers

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

[0114] Taking katG gene codon 315-M1, G463T, C(-15)T of inhA gene, A110G and G268A site mutation detection liquid chip of ndh gene as an example, for codon 315-M1, G463T, C(-15) The wild-type and mutant types of T, A110G and G268A sites design the specific primer sequence at the 3' end of the ASPE primer, and the Tag sequence at the 5' end of the ASPE primer is selected from SEQ ID NO.1-SEQ ID NO.14, correspondingly, The anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.29-SEQ ID NO.42. The specific design is shown in the following table (Table 7). The synthesis of ASPE primers, microspheres coated with anti-tag sequences, amplification primers, detection methods, etc. are as described in Ex...

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Abstract

The invention discloses a specific primer and a liquid-phase chip for katG, inhA and ndh genetic mutation detection. The liquid-phase chip mainly comprises ASPE primers, microspheres and amplification primers, wherein each ASPE primer is selected from SEQ ID NO. 15 and more than one of sequences from SEQ ID NO. 16 to SEQ ID NO. 20 at the codon 315 locus of a katG gene, SEQ ID NO. 21 and SEQ ID NO. 22 at the G463T locus of the katG gene, SEQ ID NO. 23 and SEQ ID NO. 24 at the C(-15)T locus of an inhA gene, SEQ ID NO. 25 and SEQ ID NO. 26 at the A110G locus of an ndh gene, and / or SEQ ID NO. 27 and SEQ ID NO. 28 at the G268A locus of the ndh gene. The coincidence rate of the detection result of the katG, inhA and ndh genetic mutation detection liquid-phase chip with a sequencing method is up to 100 percent; and the time required by detection is less than that required by frequently-used sequencing technology.

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 katG, inhA and ndh gene mutation detection and a liquid phase chip. Background technique [0002] The drug resistance mechanism of Mycobacterium tuberculosis to the anti-tuberculosis chemotherapy drug isoniazid is relatively complex, and most of the drug resistance of Mycobacterium tuberculosis is related to gene mutations such as katG, inhA and ndh. [0003] The katG gene (catalase-peroxidase coding gene), its N-terminal encodes peroxidase with a wide range of actions, and its C-terminal encodes catalase. This enzyme plays a role in the oxidative stress response of bacteria and is widely found in bacteria. In Mycobacterium tuberculosis, the catalase-peroxidase encoded by the katG gene converts isoniazid into a toxic form, and this activated form of isoniazid binds to a gene encoded in the mycobacterial mycolic acid bi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森吴诗扬罗小笛邓晶晶
Owner SUREXAM BIO TECH