Primer and probe of fluorescence quantitative PCR detection of superbug NDM-1 gene as well as application thereof

A super bacteria, fluorescence quantitative technology, applied in the biological field, can solve the problems such as the lack of a unified detection method kit, targeted research has not been carried out, and the biological materials and preparations are not clear, so as to control the spread of super bacteria and control people's drug use. Safe, easy-to-use operating procedures

Inactive Publication Date: 2011-08-10
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the emergence of superbugs not long ago, there have been only 3 reports of positive cases of superbugs in my country so far. The targeted research has not yet been

Method used

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  • Primer and probe of fluorescence quantitative PCR detection of superbug NDM-1 gene as well as application thereof
  • Primer and probe of fluorescence quantitative PCR detection of superbug NDM-1 gene as well as application thereof
  • Primer and probe of fluorescence quantitative PCR detection of superbug NDM-1 gene as well as application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Design and screening of primers and TaqMan probes for fluorescent quantitative PCR detection of superbug NDM-1 gene

[0029] Referring to the complete sequence of the NDM-1 gene included in GenBank (GeneBank: FN396876), select the conserved region of the NDM-1 gene, and use ABI Primer Express 3.0 real-time fluorescent quantitative PCR primer design software to design and synthesize TaqMan probes and primers. In the design of primers and probes, 2 or less degenerate bases are allowed at the same variable site. Screen the extracted candidate primers that meet the following requirements: ①The probe length (L) is between 19-28bp; ②The Tm value is between 58-60°C; ③GC% is between 25-75%; ④polyN≤4bp ; ⑤Hairpin≤4bp; ⑥Coverage >90%; ⑦The length of the amplified product is controlled between 50-150bp; ⑧The Tm value of the primer is about 10°C lower than the Tm value of the probe; ⑨Perform BLAST screening, the specificity score>L× 0.4. The optimal Tm value is set at ...

Embodiment 2

[0042] Embodiment 2, establishment of fluorescent quantitative PCR reaction system and reaction conditions of superbug NDM-1 gene

[0043] The DNA extracted from the superbug (amplified in Example 1) was diluted with sterile water in a 10-fold gradient, and used as a template for optimizing the fluorescent quantitative PCR reaction system. take 10 -4 、10 -5 、10 -6 、10 -7 、10 -8 、10 -9 Dilution, the serial number corresponds to L1, L2, L3, L4, L5, L6. Store at -70°C after aliquoting.

[0044] 1. Selection of Fluorescence Quantitative PCR Buffer

[0045] In order to compare the performance differences of fluorescent quantitative PCR reaction buffers from different manufacturers, using the same DNA template, the imported Takara reagent and the domestic Tiangen company reagent were selected for parallel comparison. The PCR reaction system is: TaqMan probe 1.0 μL (5 pmol / μL), upstream primer 1.5 μL (5 pmol / μL), downstream primer 2.5 μL (5 pmol / μL), sample DNA 5 μL, Taq DNA ...

Embodiment 3

[0073] Embodiment 3, superbug NDM-1 gene fluorescent quantitative PCR detection

[0074] 1. Establishment of standard curve

[0075] 1. Construction of pBlue-NDM-1 recombinant plasmid

[0076] The NDM-1 sequence was synthesized by Shanghai Sangon Company, and cloned into pBluescript II SK (+) (purchased from Takara Company) vector multi-cloning site between the HindIII and BamHI restriction sites to obtain the recombinant vector pBlue-NDM-1 .

[0077] 2. Standard testing

[0078] The pBlue-NDM-1 plasmid DNA was used as a template to perform fluorescent quantitative PCR detection with the superbug probe method, and a standard curve was established. The specific operation is as follows: the plasmid DNA was serially diluted 10 times to I: 1.0×10 10 Copy / μL; Ⅱ: 1.0×10 9 Copy / μL; Ⅲ: 1.0×10 8 Copy / μL; Ⅳ: 1.0×10 7 Copy / μL; Ⅴ: 1.0×10 6 Copy / μL; VI: 1.0×10 5 Copy / μL; VII: 1.0×10 4 Copy / μL; Ⅷ: 1.0×10 3 Copy / μL; IX: 1.0×10 2 Copy / μL; Ⅹ: 1.0×10 1 Copy / μL; Ⅺ: 1.0×10 0 copies / μL...

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Abstract

The invention discloses a probe and a primer in fluorescence quantitative PCR detection of a superbug NDM-1 gene as well as application thereof. The primer and the TaqMan probe are designed according to a conserved area of the superbug NDM-1 gene and used for quantitatively detecting the nucleic acid copy number of superbugs in a sample to be detected. Specially, an upstream primer of the primer has a nucleotide sequence of SEQ ID NO:1 in a sequence table, and a downstream primer of the primer has a nucleotide sequence of SEQ ID NO:2 in the sequence table; and the TaqMan probe has a nucleotide sequence of SEQ ID NO:3 in the sequence table. The invention can be used for rapidly and accurately detecting the superbug NDM-1 gene, can be used for detecting NDM-1 genes of various samples and environmental samples of a suspicious superbug disease patient and assisting the clinical diagnosis and treatment of superbug infection, has significance to control superbug propagation and guarantee human health and has wide application prospect.

Description

technical field [0001] The invention relates to a molecular biology detection method of bacteria in the field of biotechnology, in particular to primers and probes in the fluorescent quantitative PCR detection method of superbacteria NDM-1 gene. Background technique [0002] Super bacteria is a general concept that refers to bacteria that are resistant to multiple antibiotics. In the past, super bacteria were considered to be methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. The recently reported super bacteria referred to by the media refers to pan-drug-resistant bacteria containing the NDM-1 gene (Kamarasamy K, Toleman MA, Walsh TR, et al. Emergence of a new antibiotic resistance in India, Pakistan, and the UK: a prospective survey. Lancet Infect Dis, 2010, 10: 597-602). In August 2010, Lancet.Inf.Dis magazine reported that some pan-drug-resistant Gram-negative bacteria were found in India, Pakistan and the United Kingdom. -β-lactamase 1)...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 王玉飞陈泽良黄留玉杜昕颖汪舟佳宋宏彬袁静
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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