Yersinia pestis detection kit based on real-time fluorescence RPA technology and application thereof

A Yersinia pestis and kit technology, applied in the biological field, can solve the problems of long measurement time, limited field and on-site detection, etc., and achieve high sensitivity, good specificity, and broad prospects

Active Publication Date: 2021-06-04
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods can accurately detect Yersinia pestis, they are limited by long determination time (not less than several hours), expensive and sophisticated instruments (such as qPCR instruments), professional biotechnologists and high experimental environment requirements. field and field testing

Method used

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  • Yersinia pestis detection kit based on real-time fluorescence RPA technology and application thereof
  • Yersinia pestis detection kit based on real-time fluorescence RPA technology and application thereof
  • Yersinia pestis detection kit based on real-time fluorescence RPA technology and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] 1 Preparation of bacterial genome template

[0035]1.1 Use 1 mL of heat-inactivated Yersinia pestis (Yersiniapestis) bacterial liquid to extract the whole genome DNA of the bacterial liquid using the Bacterial Genomic DNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd., Cat. No. DP320), elute with 100 μL TE buffer, and use Qubit 3.0 fluorescence method was used to quantify genomic nucleic acid to obtain the genome template of Yersinia pestis (Yersiniapestis).

[0036] 1.2 Replace Yersinia pestis (Yersiniapestis) in step 1.1 with Bacillus anthraci (Bacillus anthraci), Burkholderia pseudomallei (Burkholderia apseumallei), Burkholderia mallei (Burkholderia mallei), cattle Brucella bovis, Bacillus cereus, Bacillus thuringiensis, Francisella tularensis, Salmonella typhimurium, Bacillus subtilis, sheep cloth Brucella melitensis, Escherichia coli, Vibrio vulnificus, Staphylococcus aureus, Vibrioparahaemolyticus, and Staphylococcus epidermidis, other manipulations Wi...

Embodiment 2

[0051] Embodiment 2 sensitivity analysis

[0052] In order to verify the sensitivity of the real-time fluorescent RPA reaction, the plasmid containing the target fragment (i.e. the positive plasmid prepared in step 3 of Example 1) was diluted by 10 times, so that its concentration was respectively at 10 4 ~10 -1 Between copies / μL, use the diluted plasmid as a template to perform a real-time fluorescent RPA reaction with the screened Yersinia pestis RPA primer (YPS723-F1 / R1) and probe (YPS723-1-P1), and use nucleic acid-free water as a negative Contrast, described in reaction system embodiment 1, reaction result is as follows figure 1 as shown, figure 1 Among them, A is the sensitivity fluorescence diagram, each amplification curve from top to bottom uses a positive plasmid template concentration of 10 4 -10 -1 Copy number / μL; B is the scatter diagram of independent repeatability detection, n=4 independent repeated experiments, t test, ns, not significant; *, p<0.05; **, P<...

Embodiment 3

[0054] Embodiment 3 specificity analysis

[0055]Bacillus anthraci, Burkholderia pseudomallei, Burkholderia mallei, Brucella bovis, Bacillus cereus, Bacillus thuringiensis, Tularaemia, Salmonella typhimurium, Bacillus subtilis, Brucella melitensis, Escherichia coli, wound arc The mixed genomes of 15 pathogenic bacteria, vibriovulnificus, Staphylococcus aureus, Vibrio parahaemolyticus, and Staphylococcus epidermidis, were analyzed for specificity verification, and Yersinia pestis DNA was positive control.

[0056] The specific experimental steps are: using the mixed genomes of the genomes of 15 pathogenic bacteria in step 1 in Example 1 as templates, and the Yersinia pestis RPA primers (YPS723-F1 / R1) and probes (YPS723-1- P1) Real-time fluorescent RPA reaction is carried out, as described in the reaction system embodiment 1, the genome template of Yersinia pestis is used as a positive control, the reaction conditions are the same, and the reaction results are as follows fig...

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Abstract

The invention discloses a yersinia pestis detection kit based on a real-time fluorescence RPA technology and application of the yersinia pestis detection kit. The kit package is composed of a primer pair and a probe with the name of YPS723-1-P1, wherein the primer pair is composed of single-stranded DNA with the name of YPS723-F1 and single-stranded DNA with the name of YPS723-R1. The kit disclosed by the invention can be used for rapidly and conveniently detecting yersinia pestis with high sensitivity and good specificity, and is expected to become a rapid diagnosis auxiliary tool for clinical samples.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a detection kit for Yersinia pestis based on real-time fluorescent RPA technology and its application. Background technique [0002] Plague is a severe infectious disease caused by the Gram-negative bacteria Yersinia pestis (hereinafter referred to as "Yersinia pestis"), which can cause large-scale epidemics in rodents through fleas. Humans living near plague foci can become infected through contact with infected animals or through flea bites. Plague can be transmitted from person to person through polluting aerosols, and the effective transmission distance is only 2 meters. If patients do not receive antibiotic treatment as soon as possible after symptoms appear, it may be life-threatening. In addition, Yersinia pestis has been considered as one of the typical biological warfare agents. Currently, plague has caused more than 200 million deaths worldwide. In my country, the plague...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/10C12N15/11C12R1/01
CPCC12Q1/6844C12Q1/689C12Q2521/507C12Q2563/107
Inventor 袁媛王景林宋亚军李佳欣辛文文康琳王菁李岩伟高姗
Owner ACADEMY OF MILITARY MEDICAL SCI
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