Specific primer set and detection kit for detecting drug resistance mutation gene of Mycoplasma pneumoniae

A technology for Mycoplasma pneumoniae and drug resistance mutation, which is applied in the directions of microorganism-based methods, microorganisms, recombinant DNA technology, etc., can solve the problem that the upstream primer hypoxanthine insertion site adversely improves the sensitivity, the detection sensitivity of Mycoplasma pneumoniae drug resistance is low, and the sensitivity and The specificity cannot be improved at the same time, so as to achieve the effect of good consistency, high sensitivity and good specificity

Active Publication Date: 2020-08-14
AUTOBIO DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The first object of the present invention is to solve the problem that the detection sensitivity of Mycoplasma pneumoniae drug resistance is low in the prior art; Xanthine will reduce the detection specificity, so that the sensitivity and specificity cannot be improved at the same time, and a specific primer set for detecting the drug resistance mutation gene of Mycoplasma pneumoniae is proposed

Method used

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  • Specific primer set and detection kit for detecting drug resistance mutation gene of Mycoplasma pneumoniae
  • Specific primer set and detection kit for detecting drug resistance mutation gene of Mycoplasma pneumoniae
  • Specific primer set and detection kit for detecting drug resistance mutation gene of Mycoplasma pneumoniae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Establishment of Mycoplasma pneumoniae drug-resistant mutation gene detection method

[0059] 1. Primer Design

[0060] This kit uses the 23S rRNA V region sequence of Mycoplasma pneumoniae as a template to design upstream primers (23NU, 2063MU, 2064MU), and introduces the mismatched base hypoxanthine I at the penultimate 2, 3, and 4 positions of the 3' end of the specific primer 2063MU. , the mismatched base hypoxanthine I was introduced into the penultimate 3, 4, and 5 positions of the 3' end of the specific primer 2064MU, and hypoxanthine was introduced into the penultimate position of the 3' end of its downstream primer (23D) for the first time I, to increase primer specificity. A total of 16 pairs of primers were designed during the development of this kit, and the best primer pair was selected after multiple verifications.

[0061] 2. Standard product construction: construct the wild-type and mutant plasmids containing the target gene locus, perform qu...

Embodiment 2

[0064] Embodiment 2 Preparation and use method of kit of the present invention

[0065] The composition of kit of the present invention:

[0066] 1. The primer sequence group that is used to detect Mycoplasma pneumoniae mutant gene, described primer group is shown in SEQ ID No.1 by wild-type upstream primer sequence, A2063G mutant type upstream primer sequence is shown in SEQ ID No.2, A2064G mutation The sequence of the type upstream primer is shown in SEQ ID No.3, and the sequence of the universal downstream primer is shown in SEQ ID No.4.

[0067] 2. The PCR reaction solution can be a composition known to those skilled in the art. The recommended composition of the present invention is: 25 μl of PCR buffer UltraSYBR Mixture (With ROX), (UltraSYBR Mixture, purchased from Beijing Kangwei Century Biotechnology Co., Ltd., article number: CW2601M) . Nucleic acid-free water 22 μl, template 2 μl.

[0068] The method for using the kit of the present invention:

[0069] 1. Extrac...

Embodiment 3

[0095] Embodiment 3 kit sensitivity and specificity verification experiment

[0096] (1) Standard curve drawing

[0097] Use specific primers (SEQ ID No.2 2063MU, SEQ ID No.3 2064MU) and non-specific primers (SEQID No.1 23NU) to amplify the corresponding mutant standard respectively (do 3 holes for each reaction, Ct=MeanCt±SD ), with the logarithm value of the standard substance concentration as the abscissa, and the corresponding Ct value as the ordinate, the corresponding specificity and non-specificity standard curves are obtained, such as figure 1 and figure 2 As shown, the standard curves almost overlap, indicating that the introduction of non-matching base (I) in the specific primer sequence does not affect the amplification efficiency of the primers, the template amount has a good correlation with the Ct value, and the template can be quantified. in, figure 1 Middle standard curve 1: The straight line is the specific standard curve obtained by amplifying the A2063G ...

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Abstract

The invention discloses a specific primer pair for detecting drug resistance mutation gene of mycoplasma pneumonia. The primer pair is designed by using mycoplasma pneumonia 23S rRNA V-zone wild-type sequence and A2063G and A2064G mutant-type sequence as template. The primer pair is composed of wild-type upstream primer sequence as shown in SEQ ID No. 1, A2063G mutant-type upstream primer sequence as shown in SEQ ID No.2, A2064G mutant-type upstream primer sequence as shown in SEQ ID No.3, and universal downstream primer sequence as shown in SEQ ID No. 4. The detection kit has very high sensitivity and specificity, and can quantify mutant DNA and wild template DNA so as to effectively distinguish various genotypes and calculate corresponding ratio of various genotypes. Dynamic monitoring of genotypes and content changes of microflora in the body of a patient with clinical mycoplasma pneumoniae infection can be realized. The detection kit is beneficial to clinical researches on drug resistance mechanism and progress of mycoplasma pneumonia and is convenient for observation of clinic therapy effect.

Description

technical field [0001] The invention relates to a specific primer for detecting drug-resistant mutant genes of Mycoplasma pneumoniae, in particular to specific primers for detecting drug-resistant A2063G and A2064G mutant sequences of Mycoplasma pneumoniae and a detection kit prepared therefrom. Background technique [0002] The main medicines for the clinical treatment of Mycoplasma pneumoniae infection are macrolides, tetracyclines and quinolones. Since the first macrolide-resistant strain of Mycoplasma pneumoniae was isolated in Japan in 2000, drug-resistant Mycoplasma pneumoniae infections have been reported all over the world. Especially in Asia, the phenomenon of drug resistance is the most serious. Both China and Japan have repeatedly reported that the drug resistance rate exceeds 90%. It is imminent to develop a detection kit for detecting the drug resistance of Mycoplasma pneumoniae. In recent years, many countries have reported that the same patient was simultaneou...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/6858C12Q1/06C12N15/11C12R1/35
CPCC12Q1/6858C12Q1/689C12Q2600/106C12Q2600/156C12Q2531/113C12Q2535/125C12Q2545/113C12Q2563/107
Inventor 郭东星辛德莉胡文娟侯安存申昆玲
Owner AUTOBIO DIAGNOSTICS CO LTD
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