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PCR primer pair and kit for rodent-borne disease pathogenic microorganisms and application thereof

A technology of pathogenic microorganisms and primer pairs, applied in the field of microbiology, can solve problems such as complex operation, low sensitivity, and high cost

Inactive Publication Date: 2019-09-10
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional detection methods such as separation and culture, serological detection, etc. have low throughput and low sensitivity, and various high-throughput detection methods based on nucleic acid molecular detection technology have emerged as the times require, such as Cai Jing et al. [5] The multiple real-time fluorescent probe method was used to realize the typing detection of three pathogenic types of Borrelia burgdorferi. The detection was completed by designing a pair of universal primers and three specific probes, and the lowest detection limit could reach 5 copies / μl. The limited number of detection channels limits the number of detected pathogens; Jana et al. [6] A method for detecting tick-borne pathogens by combining DNA microarray and PCR technology was established. Based on the 16S variable region, it simultaneously detected Coxia beinii, Rickettsia and Francisella tularensis with detection limits of 10 copies / μl, respectively. 100copies / μl, 1copies / μl, this method is complex and expensive

Method used

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  • PCR primer pair and kit for rodent-borne disease pathogenic microorganisms and application thereof
  • PCR primer pair and kit for rodent-borne disease pathogenic microorganisms and application thereof
  • PCR primer pair and kit for rodent-borne disease pathogenic microorganisms and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1: Specific gene screening and primer design

[0108] On the microbial data analysis cloud platform (https: / / analysis.mypathogen.org), upload the target microorganisms Leptospira interrogans (GenBank accession number: AE010300.2), Rickettsia moschii (GenBank accession number: AE017197.1) ), Orientia tsutsugamushi (GenBank accession number: AM494475.1), Anaplasma phagocytophilum (GenBank accession number: CP000235.1), Francisella tularensis (GenBank accession number: AJ749949.2), Coxia basilica (GenBank accession number: AE016828.3) and Bartonella (GenBank accession number: BX897699.1) genome sequences, compared with other taxonomically similar microbial genome sequences and the gene coding sequences (coding sequences, CDS), select the "special marker gene" workflow to run, and obtain the specific gene of the target microorganism, wherein, the specific gene of Leptospira interrogans is the 99185th-100006th in the genome base sequence (referred to as the Lep speci...

Embodiment 2

[0116] Embodiment 2: establishment and optimization of multiplex PCR system

[0117] First establish a single-plex PCR reaction, the reaction system: 1 μl (1 μmol / L) of upstream and downstream chimeric primers CF and CR for one of the 7 kinds of microorganisms, 1 μl (10 μmol / L) of upstream and downstream general primers GF and GR (10 μmol / L), 2×master mix 12.5 μl, template 2 μl, ddH 2 O 6.5 μl. Reaction conditions: 95°C for 5 minutes; Stage 1: 95°C for 30s, 60°C for 1min, 72°C for 1min, cycle 10 times; Stage 2: 95°C for 30s, 69°C for 1min, 72°C for 1min, cycle 10 times; Stage 3: 95°C 30s, 45°C for 1min, 72°C for 1min, cycle 20 times; 72°C for 10min. Set up gradient PCR to optimize primer concentrations. On the basis of a single reaction, increase the primers sequentially, increase the number of reactions, and optimize the primer concentration, extension time and cycle number. The product was detected by QIAxcel capillary electrophoresis (the same below).

[0118] Taking t...

Embodiment 3

[0123] Embodiment 3: multiple PCR system specificity and sensitivity detection

[0124] The present invention designs 7 plasmid standard products in total, which are the recombinant plasmids containing the respective specific genes formed by inserting the respective specific genes used in Example 1 into T vectors, specifically: the specific genes of Ap, Cb, Bar, and Lep are inserted independently In the pUC57-Simple plasmid, the specific genes of Ot, Ft, and Rt were independently inserted into the pMD-19T-Simple plasmid to form respective recombinant plasmids. The mixed plasmids and mixed plasmid standard products mentioned in the present invention all refer to the mixture of these 7 plasmid standard products.

[0125] According to the conversion formula of copy number concentration: copy number concentration (copies / μl)=[DNA concentration (ng / μl) / base number×660]×6.02×10 23 , Dilute the plasmid standard by 10 times to 100-10 5 copies / μl.

[0126] (1) Multi-primer single-te...

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PUM

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Abstract

The invention discloses a PCR primer pair for rodent-borne disease pathogenic microorganisms. The primer pair is used for amplifying specific genes of leptospira interrogans, Rickettsi Mooseri, Orientia tsutsugamushi, anaplasma phagocytophilum, francisella tularensis, Coxiella burnetii and Bartonella. By adopting the PCR primer pair, on the basis of the TSP principle, the method for high-sensitiveand specific independent or combined detection of the seven kinds of pathogene.

Description

technical field [0001] The invention belongs to the field of microbiology, and in particular relates to a multiplex PCR detection method for seven kinds of mouse-borne pathogenic microorganisms, as well as reagents and applications thereof. Background technique [0002] Rat-borne diseases refer to zoonotic infectious diseases caused by rodents and transmitted between humans / animals, also known as zoonoses. Rodents can transmit pathogens such as certain viruses, bacteria, rickettsia, protozoa and worms to humans and domestic animals, and are the reservoir hosts of various zoonotic pathogens. In recent years, with the accelerated process of globalization, human activities have increased, and the mobility of people and things has increased. The epidemic area of ​​rat-borne diseases has also continued to expand. Many new rat-borne diseases have emerged and some old infectious diseases have reemerged. [1-3] , such as leptospirosis caused by Leptospira interrogans (Lep) and endem...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689C12Q2600/16Y02A50/30
Inventor 栗冬梅刘起勇张燕君宋秀平康央
Owner ICDC CHINA CDC
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