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Target sequence and kit for detection of metallo-beta-lactamase-1 gene in New Delhi
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A technology of β-lactamase and kit, which is applied in the field of biochemistry, can solve the problems that cannot meet the needs of identification of pathogenic microorganisms and guidance of medication, etc., achieve reasonable design of technical solutions, avoid false positive results, and ensure detection specificity and sensitivity Effect
Active Publication Date: 2016-03-02
SHANGHAI XINGYAO MED TECH DEV CO LTD +1
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[0003] The traditional clinical detection of multidrug-resistant bacteria of this kind of antibiotics is the method of isolation and culture combined with drug susceptibility identification. It takes more than a week to get the test results at the fastest, which is far from meeting the clinical needs of pathogenic microorganism identification and medication guidance.
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Embodiment 1
[0039] Example 1: Preparation of NDM-1 gene fluorescent PCR detection kit
[0040] (1) Primer probe synthesis
[0041] Entrust a commercial sequence synthesis company (Shanghai Sangon Bioengineering Technology Service Co., Ltd.) to synthesize the following sequences:
[0042] The upstream primer is 5'-ggCggggATTgCgACTTATg-3', as shown in SEQ ID No: 2;
[0043] The downstream primer is 5'-AATACCTTgAgCgggCCAAAg-3', as shown in SEQ ID NO: 3;
[0044] The fluorescent probe is 5'-AATgCgTTgTCgAACCAgCTTgCC-3' (5' labeled FAM, 3' labeled TAMRA or BHQ-1), as shown in SEQ ID No: 4.
[0045] Dissolve them in sterile double-distilled water to a concentration of 25 μM, and store at -20°C.
[0046] (2) Preparation of NDM-1 gene PCR buffer
[0047] Contains Tris-HCl (pH8.3) 14.2mM, KCl71.4mM, gelatin 0.14mg / ml, dATP, dGTP, dCTP, dUTP each 0.29mM, MgCl 2 5 mM, 5 μM each of upstream and downstream primers (SEQ ID No: 2 and 3), aliquoted according to 672 μl / tube.
[0048] (3) Preparation of...
Embodiment 2
[0062] Example 2: Application of NDM-1 gene fluorescent PCR detection kit
[0063] (1) Sample processing
[0064] Take 20 cases of collected clinical samples or enrichment solution, put them in a centrifuge tube and centrifuge at 13,000rpm for 6 minutes, carefully discard the supernatant with a pipette (5μl residual liquid can be kept to prevent the precipitation from being sucked away), and add 1ml of Physiological saline, vortex to mix, repeat centrifugation and discard the supernatant. Add 50 μl of DNA extraction solution to the pellet, vortex to mix, keep at 100°C for 10 minutes, centrifuge at 13,000 rpm for 2 minutes, and take the supernatant for fluorescent PCR amplification detection. The negative and positive controls of the kit are processed in the same way as the sample solution.
[0065] (2) Amplification detection
[0066] According to the number of samples to be tested n+2 (n=20), take the kit PCR buffer 21 μl×(n+2), fluorescent probe 3 μl×(n+2), Taq enzyme 2 μ...
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Abstract
The invention discloses a target sequence and a kit for detecting New Delhimetallo-bata-lactamase-1 (NDM-1) gene by a fluorescent polymerase chain reaction (PCR) technology. A region of the target sequence SEQ IDNo:1 for the NDM-1 gene is amplified by a pair of primers, an amplification product is detected by fluorescent probes, and the existence of the NDM-1 gene is judged according to a template fluorescent signal and a cycle threshold. The kit comprises the following components: a PCR buffer solution, fluorescent probes, Taq DNA polymerase, a negative control, a positive control and a nucleic acid extracting solution. The using method for the kit comprises two steps of sample treatment and amplification detection. The kit is easy and convenient to operate, high in sensitivity and high in specificity, and can be widely applied to quickly detecting various antibiotic resistant pathogenic bacteria caused by the NDM-1 gene clinically.
Description
technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a target sequence and a kit for detecting New Delhi metallo-beta-lactamase-1 (NDM-1) gene by using fluorescent PCR technology. Background technique [0002] In 2010, a new type of bacterial variant gene that appeared in South Asian countries such as India and Pakistan may spread globally. Bacteria with this gene are resistant to most antibiotics including cephalosporins, carbapenems, and aminoglycosides. Drug resistance (currently only sensitive to two antibiotics, tijiacycline and polymyxin), so medical experts named this gene New Delhi metallo-β-lactamase-1 (New Delhimetallo-β-lactamase1, referred to as NDM- 1) Gene, which originally existed on the plasmid of bacteria such as Escherichia coli, and the enzyme produced by expression can decompose the above-mentioned antibiotics. Resistance to antibiotics (YongD, et al. Antimicrobial Agents and Chemotherapy, 2009...
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