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Fluorescent quantitative PCR kit for detecting vancomycin-resistant vanA gene of enterotoxin

A vancomycin-resistant, fluorescence quantitative technology, applied in the field of bioengineering, can solve problems such as difficult clinical treatment, increased infection incidence and mortality, and achieve the effect of optimizing response parameters

Pending Publication Date: 2020-04-24
SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Enterotoxin, as an opportunistic pathogen, has continuously increased the incidence of infection and mortality in recent years, and has become an important pathogen of nosocomial infection, which has caused many difficulties in clinical treatment.

Method used

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  • Fluorescent quantitative PCR kit for detecting vancomycin-resistant vanA gene of enterotoxin
  • Fluorescent quantitative PCR kit for detecting vancomycin-resistant vanA gene of enterotoxin
  • Fluorescent quantitative PCR kit for detecting vancomycin-resistant vanA gene of enterotoxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: S1, extract GenBank: KF052036.1

[0054]

[0055]

[0056] S2, the following sequence is designed by software (Beacon Designer8.20):

[0057] SEQ ID NO: 1 upstream primer F: 5'-GCTACGTTTACCTATCCTG-3'

[0058] SEQ ID NO: 2 downstream primer R: 5'-CAGCCTGCTCAATTAAGA-3'

[0059] SEQ ID NO: 3TaqMan probe sequence P: 5'-FAM-TTGCTGTCATATTGTCTTGCCGATTC-BHQ-3'; the reporter fluorescent group labeled at the 5' end of the probe and the quencher group labeled at the 3' end can be selected according to the fluorescent PCR equipment, etc. The specific situation is selected separately, and the detection of fluorescent RT-PCR.

[0060] (1) Take the RT-PCR reaction solution, hot-start Taq enzyme and probe according to the number of detection samples n (n=the number of samples to be tested+2) and mix them in a centrifuge tube, vibrate on a vortex shaker, and Aliquot the tubes and cap the tubes for later use.

[0061] (2) Now add the negative control solution into an...

Embodiment 2

[0071] Embodiment 2: standard curve test

[0072] Take the positive plasmid in the kit, determine its concentration to be 178.8ng / μL, and carry out 10-fold gradient dilution with DEPC water, and dilute 6 gradients in total, and perform fluorescent RT-PCR detection on each dilution respectively to obtain Its slope and correlation coefficient R2 value.

[0073] Experimental results: For the standard curve of the plasmid, see figure 1 , the slopes are -3.323, respectively, according to the formula E (amplification efficiency) = 10-1 / slope, the amplification efficiency converted into a percentage ((E-1) × 100%) is 103.1%, between 90% to 110% between 0.99 and 1, indicating that the amplification efficiency of the method is good; the R2 values ​​are 0.999, respectively, between 0.99 and 1, indicating that the method has high reliability.

Embodiment 3

[0074] Embodiment 3: specificity test

[0075] Take Escherichia coli standard strain, Salmonella standard strain, Staphylococcus aureus standard strain, Campylobacter standard strain DNA each 2 μL as the template for the specificity detection of fluorescent RT-PCR method, and set the plasmid as positive control and blank plasmid as negative As a control, DEPC water was used as a blank control group.

[0076] RT-PCR amplification conditions: 5 min at 42°C, pre-denaturation at 95°C for 10 s, cycled 40 times according to the following parameters: denaturation at 94°C for 15 s, fluorescence collection at 59°C for 30 s. Fluorescence detection at 59°C, detection channel: FAM.

[0077] The results of fluorescent RT-PCR showed that enterotoxins containing vanA gene amplified curves, while other bacteria did not have S-type amplification curves. At the same time, DEPC water blank control and blank plasmid negative control were both established, indicating that the method has good spec...

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Abstract

The invention relates to the technical field of bioengineering, and especially relates to a fluorescent quantitative PCR kit for detecting a vancomycin-resistant vanA gene of enterotoxin. SEQ ID NO:1upstream primer F:5'-GCTACGTTTACCTATCCTG-3', SEQ ID NO:2 downstream primer R:5'-CAGCCTGCTCAATTAAGA-3' and SEQ ID NO:3 TaqMan probe sequence P:5'-FAM-TTGCTGTCATATTGTCTTGCCGATTC-BHQ-3' are designed through software. Fluorescent RT-PCR detection is carried out, an RT-PCR reaction liquid, a hot start Taq enzyme and a probe are taken and mixed into a centrifugal tube according to the number n (n = (thenumber of samples to be detected) + 2) of detection samples, the centrifugal tube is oscillated on a vortex oscillator, sub-packaging is carried out according to each tube, each tube is covered witha tube cover for later use, a negative control liquid is added into a sub-packaging tube, and DNA of each sample is taken, and is added into a corresponding reaction tube; and finally, a positive control is taken out and is added into another reaction tube, and all the reaction tubes are labeled and centrifuged, and are taken out of a fluorescent PCR instrument. The kit can rapidly detect the enterococcus vancomycin VanA drug-resistant gene, and provides guidance for drug therapy after super bacterial infection.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a fluorescent quantitative PCR kit for detecting the vancomycin-resistant vanA gene of enterotoxin. Background technique [0002] Enterotoxins are normal flora in the digestive tract of humans and animals, and are widely distributed in the natural environment. Enterotoxin, as an opportunistic pathogen, has continuously increased the incidence of infection and mortality in recent years, and has become an important pathogen of nosocomial infection, which has caused many difficulties in clinical treatment. Due to the strong and thick cell walls of enterotoxins, they are inherently resistant to multiple antibacterial drugs, such as β-lactams, low-concentration aminoglycosides, erythromycin, clindamycin and other antibacterial drugs. With the widespread clinical use of new antimicrobial drugs, driven by drug pressure, enterobacteria have produced many new acquired drug-resis...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/04
CPCC12Q1/689C12Q1/6851C12Q2600/166C12Q2531/113C12Q2563/107C12Q2545/113
Inventor 孙冰清姜芹黄士新张文刚曹莹顾欣商军王建严凤张浩然潘娟田恺吴剑平黄家莺
Owner SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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