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Fluorescent quantitative polymerase chain reaction (PCR) kit for Acinetobacter baunannii/calcoaceticus complex OXA-23 mutant gene

A technology for OXA-23 and mutant genes, applied in the field of drug resistance gene detection kits, can solve the problems of infection control and clinical treatment difficulties, endangering the lives of patients, etc.

Active Publication Date: 2012-07-25
TAIZHOU MUNICIPAL HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The emergence of multidrug-resistant Acinetobacter baumannii complex calcium acetate has brought great difficulties to hospital infection control and clinical treatment, because most of the infected patients are elderly patients, critically ill patients and patients with weak body resistance, such as Failure to select antibiotics sensitive to Acinetobacter Baumann complex calcium acetate for treatment in time will endanger the lives of patients

Method used

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  • Fluorescent quantitative polymerase chain reaction (PCR) kit for Acinetobacter baunannii/calcoaceticus complex OXA-23 mutant gene
  • Fluorescent quantitative polymerase chain reaction (PCR) kit for Acinetobacter baunannii/calcoaceticus complex OXA-23 mutant gene
  • Fluorescent quantitative polymerase chain reaction (PCR) kit for Acinetobacter baunannii/calcoaceticus complex OXA-23 mutant gene

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Experimental program
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Effect test

Embodiment 1

[0018] Example 1: Determination of the new sequence of the drug-resistant gene OXA-23 of Baumann's complex Acinetobacter calcium acetate

[0019] 1. Source of strains: From October 2009 to May 2011, we collected 32 strains of Acinetobacter calcoacetate baumannii complex that were highly resistant to antibiotics (especially resistant to imipenem). The 32 specimens were derived from the patient's sputum.

[0020] 2. Design of OXA-23 primers: the present invention designs a pair of specific primers for class D β-lactamase resistance gene OXA-23. The sequences of the primers are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0021] 3. Amplification of OXA-23 gene: with above-mentioned 32 strains of Baumann's complex Acinetobacter calcium acetate lysate as template, carry out PCR amplification, amplification system is (25ul system): template DNA (bacteria lysate) 2ul, 2ul each of primers 1 and 2, TaqE: 0.25ul (0.5U), 10XBuffer 2.5ul, dNTP 2ul, with ddH 2 O make up to 25ul. The reacti...

Embodiment 2

[0029] Example 2: Preparation of a real-time fluorescent quantitative PCR kit for detecting the new sequence of OXA-23

[0030] 1. Primer design and probe synthesis

[0031] The sequence of SEQ ID NO: 3 obtained according to Example 1 above. Probes and primers (synthesized by Shanghai Jikang Company) were designed using ABI primer Express 2.0 software. See SEQ ID NO: 4 for the sequence of the upstream primer, see SEQ ID NO: 5 for the sequence of the downstream primer, and see SEQ ID NO: 6 for the sequence of the probe. The 5' end of the probe is labeled with a fluorescent chromophoric group FAM, and the 3' end is connected with a fluorescent quencher group BHQ.

[0032] 2. Standard DNA template preparation

[0033]Using the upstream and downstream primers described in the previous step, a 200bp fragment was amplified using the above-mentioned AB37 strain as a template, and the PCR product was purified (QIAgen, German) and connected to the PGM-T cloning vector and transforme...

Embodiment 3

[0039] Embodiment 3 in vitro detection experiment

[0040] 1. Sensitivity test

[0041] The standard DNA template was serially diluted 10 times, so that the concentration range of the standard plasmid was 10 8 ~10 2 copies / μl, as the standard of the fluorescence quantification kit. Fluorescent quantitative PCR reaction conditions, 25 μl reaction system: 12.5 μl 2×TaqMan Universal PCR Master Mix (ABI Company, USA), 10 nM each of the upstream and downstream primers (the sequences of the upper and lower primers are Sequence NO.4 and Sequence NO.5, respectively), 400nM probe (probe sequence is Sequence NO.6), 2μl gradient diluted standard DNA template, add ddH 2 0 to 25 μl. The reaction program was pre-denaturation at 95°C for 10 min, followed by 45 cycles: 95°C for 40 s, 60°C for 1 min. Sterile water was used as a negative control.

[0042] See the test results figure 1 , Sensitive analysis shows that a minimum of 100copies / reaction can be detected. This experiment was re...

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Abstract

The invention discloses a fluorescent quantitative polymerase chain reaction (PCR) kit and method for an Acinetobacter baunannii / calcoaceticus complex OXA-23 mutant gene. Special oligonucleotide primers and probes are designed according to sequences of the mutant of the newly screened D-type beta-lactamase drug-resistant gene OXA-23 of the Acinetobacter baunannii / calcoaceticus complex. The fluorescent quantitative PCT kit can be used for detecting the Acinetobacter baunannii / calcoaceticus complex.

Description

technical field [0001] The invention relates to a detection kit and a method for drug resistance genes. Specifically, according to the newly screened OXA-23 gene mutated Acinetobacter baumannii complex calcium acetate strain, the present invention designs a fluorescent quantitative PCR kit for detecting OXA-23 mutation Acinetobacter baumannii complex calcium acetate. The invention also relates to its detection method. Background technique [0002] Acinetobacter is a class of non-fermenting, strictly aerobic, Gram-negative bacilli that are ubiquitous in nature. Acinetobacter can be divided into at least 32 genotypes, among which Acinetobacter baumannii, Acinetobacter calcium acetate, Acinetobacter genotype 3 and 13TU are difficult to distinguish through conventional phenotypic identification, collectively known as the Baumann complex Acinetobacter calcoacetate complex. [0003] Acinetobacter baumannii is the most common gram-negative bacillus in the genus Acinetobacter, wi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/22C12N15/31C12Q1/68C12Q1/06G01N21/64
Inventor 王冬国王海宝梁勇戚永孝
Owner TAIZHOU MUNICIPAL HOSPITAL
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