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Primer combination for simultaneously identifying 8 kinds of cattle pathogens and GeXP detection method

A combination of primers and pathogens technology, applied in the direction of microbial-based methods, biochemical equipment and methods, microbial measurement/testing, etc., can solve problems such as incorrect results, time-consuming and labor-intensive problems

Inactive Publication Date: 2016-12-07
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the main methods used for the differential diagnosis of these bovine infectious diseases include pathogen isolation and identification and serological tests, etc., but these methods are often limited by the freshness of clinical disease materials, the degree of contamination or the titer of serum, resulting in incorrect results and time-consuming Power consumption, there are certain limitations in practical application
With the advancement of molecular biology, molecular biology diagnostic methods developed on the basis of PCR technology have been widely used in the detection and diagnosis of infectious diseases, including PCR, fluorescent PCR and LAMP, etc., but these methods can only be used for single or 2 to 2 4 kinds of pathogens are detected, and it is impossible to realize high-throughput detection of multiple pathogens at the same time in a true sense

Method used

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  • Primer combination for simultaneously identifying 8 kinds of cattle pathogens and GeXP detection method
  • Primer combination for simultaneously identifying 8 kinds of cattle pathogens and GeXP detection method
  • Primer combination for simultaneously identifying 8 kinds of cattle pathogens and GeXP detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0153] Embodiment 1, design and preparation of primer combination

[0154] A large number of sequence analyzes and comparisons were carried out to obtain several primers for identifying 8 bovine pathogens of FMDV, BTV, VSV, BVDV, BRV, ETEC, IBRV and PPRV. Preliminary experiments were carried out on each primer to compare performances such as sensitivity and specificity, and finally 8 pairs of specific primers for identifying 8 kinds of bovine pathogens were obtained. Each specific primer pair, forward primer and reverse primer, consists of a targeting segment and a universal primer segment, with the universal primer segment located 5' to the targeting segment.

[0155] The primer pair used to identify FMDV consists of the following two primers (5'→3'):

[0156] FMDV-F (Sequence 1 of the Sequence Listing): AGGTGACACTATAGAATA GCCGTGGGACCATACAGG;

[0157] FMDV-R (Sequence 2 of the Sequence Listing): GTACGACTCACTATAGGGA AAGTGATCTGTAGCTTGGAATCTC.

[0158] The underlined part...

Embodiment 2

[0196] Embodiment 2, specificity

[0197] 1. Single template experiment

[0198] 1. Extract the total RNA of the sample to be tested and reverse transcribe it into cDNA. The samples to be tested are: FMDV type O inactivated virus, BTV type 4 inactivated virus, VSV NJ type inactivated virus, BVDV reference strain Oregon CV24 strain (BVDV-1 type), BRV reference strain NCDV, PPRV vaccine strain , IBRV virus.

[0199] 2. Extract the genomic DNA of the sample to be tested. The samples to be tested were ETEC reference strain 1676.

[0200] 3. Using the cDNA obtained in step 1 and the genomic DNA obtained in step 2 as templates, GeXP multiplex PCR was performed using the primer combination in Example 1.

[0201] Multiplex PCR reaction system (20 μL): template 1 μL, Genome Lab GeXP Starter Kit 5×buffer 4 μL (the buffer contains universal primers, and the universal primers are primer A shown in sequence 25 of the sequence listing and primers shown in sequence 26 of the sequence lis...

Embodiment 3

[0219] Example 3, universality

[0220] 1. Extract the total RNA of the sample to be tested and reverse transcribe it into cDNA. The samples to be tested are: FMDV type O inactivated virus, FMDV type A inactivated virus, FMDV Asia type I inactivated virus, BTV type 4 inactivated virus, BTV type 8 inactivated virus, BTV type 9 inactivated virus, BTV Type 15 inactivated virus, BTV type 17 inactivated virus, BTV type 18 inactivated virus, VSV NJ type inactivated virus, VSV IND type inactivated virus, BVDV reference strain Oregon CV24 strain (BVDV-1 type), BVDV reference strain Strain NADL strain (BVDV-1 type), BVDV reference strain Yak strain (BVDV-1 type), BVDV strain GX-BVDV1, BVDV strain GX-BVDV2, BVDV strain GX-BVDV3, BVDV strain GX- BVDV4, BVDV strain GX-BVDV5, BVDV strain GX-BVDV6, BVDV strain GX-BVDV7, BVDV strain GX-BVDV8, BVDV strain GX-BVDV9, BVDV strain GX-BVDV10, BVDV strain GX-BVDV11 , BVDV strain GX-BVDV12, BVDV strain GX-BVDV13, BVDV strain GX-041, BRV reference ...

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Abstract

The invention discloses a primer combination for simultaneously identifying 8 kinds of cattle pathogens and a GeXP detection method. The primer combination provided by the invention consists of a primer pair I, a primer pair II, a primer pair III, a primer pair IV, a primer pair V, a primer pair VI, a primer pair VII and a primer pair VIII. The invention also provides the GeXP detection method for simultaneously identifying foot and mouth disease viruses, blue tongue viruses, vesicular stomatitis viruses, bovine viral diarrhoea viruses, bovine rotaviruses, enterotoxigenic escherichia coli, infectious bovine rhinotracheitis viruses and peste des petits ruminants viruses. The GeXP detection method built by the invention can be used for simultaneously identifying 8 kinds of cattle infectious disease pathogens. The method has the characteristics of high flux, specificity and high sensitivity, and can be used for the cattle disease epidemiology monitoring and the emergent epidemic situation identification and diagnosis, and guarantees the healthy development of cattle rearing industry.

Description

technical field [0001] The invention relates to a combination of primers and a GeXP detection method for simultaneously identifying 8 kinds of bovine pathogens. Background technique [0002] At present, my country has 138 million cattle on hand, and its beef output has reached 6.759 million tons, making it the fourth largest beef producer in the world. In recent years, the cattle breeding industry in Guangxi has also developed very rapidly. It has the fifth largest cattle herd in the country. Among them, the number of buffaloes on hand has reached 4.5 million, accounting for one-fifth of the total number of buffaloes in the country, ranking first in the country and second in the world. With the development of the cattle industry, the incidence of cattle infectious diseases is also increasing year by year, which has become an important factor restricting the development of the cattle industry. The specific manifestations are: old diseases are still prevalent, and new serotype...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12Q1/10C12N15/11C12R1/93C12R1/19
CPCC12Q1/686C12Q1/701C12Q2600/16C12Q2537/143C12Q2565/125C12Q1/689C12N15/11
Inventor 谢芝勋范晴谢志勤邓显文谢丽基黄莉罗思思黄娇玲张艳芳曾婷婷王盛刘加波庞耀珊
Owner GUANGXI VETERINARY RES INST
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