A detection kit for Yersinia pestis based on real-time fluorescent RPA technology and its application

Abstract

The invention discloses a Yersinia pestis detection kit based on real-time fluorescence RPA technology and its application. The kit is a complete set of reagents, consisting of a primer pair and a probe named YPS723‑1‑P1. The primer Pairs consist of single-stranded DNA with the designations YPS723‑F1 and YPS723‑R1, respectively. The kit of the invention can detect Yersinia pestis quickly, portablely, with high sensitivity and good specificity, and is expected to become a rapid diagnostic 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...

Examples

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...

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...

Examples

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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