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Multi-RT-PCR detection primer set for TGEV, PEDV, SADS-CoV and PDCoV

A technology of RT-PCR and TGEV-R, which is applied in DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc. It can solve the problems of high requirements for equipment and testing personnel, high cost, and many influencing factors , to achieve high sensitivity, good stability and good repeatability

Pending Publication Date: 2019-09-20
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ELISA method in the detection of serological technology is widely used in the detection and identification of pathogens. The advantages of this method are good specificity and high sensitivity. The disadvantage is that there are many influencing factors and high cost.
In addition, indirect immunofluorescence and immunohistochemistry are also used in the differential diagnosis of pathogens. The detection principles of these two methods are similar, and they have high specificity and sensitivity. However, the requirements of these two methods for equipment and testing personnel very high

Method used

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  • Multi-RT-PCR detection primer set for TGEV, PEDV, SADS-CoV and PDCoV
  • Multi-RT-PCR detection primer set for TGEV, PEDV, SADS-CoV and PDCoV
  • Multi-RT-PCR detection primer set for TGEV, PEDV, SADS-CoV and PDCoV

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] 1. Primer design

[0068] Reference and comparison of the full gene sequence of TGEV in GenBank (Accession: KP202948, Accession: KX083668, Accession: HM776941, Accession: KX499468, Accession: HQ462571, Accession: DQ443743, Accession: KT696544, Accession: FJ755618, Accession: EU674:218, Accession: FJ755618, Accession: EU674:218, Accession: DQ811788), the full gene sequence of PEDV (Accession: KY963963, Accession: KX981440, Accession: KR610994, Accession: KU297956, Accession: MG781192, Accession: KJ623926, Accession: KT941120, Accession: AF353511, Accession: KP890336), Accession: AF353511, Accession: KP890336), Accession: 70 The full gene sequence of SADS-CoV (Accession: MF769443, Accession: MF769442, Accession: MF769418, Accession: MF769416, Accession: MF769417, Accession: MG557844, Accession: MF094684, Accession: MF094683, CoV: MF094682, Accession: PD807) and (Accession: KY926512, Accession: MG837131, Accession: MG242026, Accession: KR131621, Accession: MF431743, Access...

Embodiment 2

[0074] Example 2 Acquisition of pMD-19-T-PEDV, pMD-19-T-TGEV, pMD-19-T-SADS and pMD-19-T-PDCoV positive recombinant plasmids

[0075] (1) Extraction of viral genome RNA

[0076] Porcine transmissible gastroenteritis virus (TGEV, CN12 strain), porcine epidemic diarrhea virus (PEDV, CH / GDGZ / 2012 strain), porcine T-type RNA of coronavirus (PDCoV, Ch-A strain) and porcine acute diarrhea syndrome coronavirus (SADS-CoV, HN17 strain), and control viruses (Seneca Valley virus (SVV) HN16 strain, porcine PRRS virus ( RNA of PRRSV) YA strain and atypical swine fever virus (APPV) APPV / CN / HUANAN / 201801 strain. The specific operation steps are carried out according to the kit.

[0077] (2) Reverse transcription of template RNA

[0078] Construct a 20 μL RNA reverse transcription reaction system, and establish RNA reverse transcription reaction parameters at the same time. The specific reaction system is: 5×M-MLV Buffer 4.0 μL, dNTP Mixture 1.0 μL, Random primer 1.0 μL, RNase inhibitor 0.5...

Embodiment 3

[0098] Example 3 Establishment and optimization of multiple RT-PCR reaction systems for TGEV, PEDV, SADS-CoV and PDCoV

[0099] A 50 μL initial multiple RT-PCR reaction system (Table 2) and reaction parameters (Table 3) were constructed, and the annealing temperature, TaKaRa Taq enzyme concentration, dNTP, and the primer concentration and ratio of the multiple RT-PCR reaction system were optimized in turn, and the obtained The results were detected by agarose gel (mass volume ratio 1.2%) electrophoresis;

[0100] Table 2 initial multiplex RT-PCR reaction system

[0101] System components Content (volume / μL) 10×Buffer 5.0 2.5mM dNTPs 4.0 Primer Mixture F 2.0 Primer Mixture R 2.0 5U / μL TaKaRa Taq 1.0 template 4.0 RNase Free ddH 2 o

32 total 50

[0102] Note: In the above system, the final concentration of a single primer is 0.1 μmol / L;

[0103] Table 3 initial multiplex RT-PCR reaction parameters

[0104] ...

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Abstract

The invention relates to the technical field of biotechnology, in particular to a multi-RT-PCR detection primer set for TGEV, PEDV, SADS-CoV and PDCoV. The nucleotide sequence of the multi-RT-PCR detection primer set for TGEV, PEDV, SADS-CoV and PDCoV is shown as SEQ ID NO. 1-8. The invention also provides a kit comprising the primer set. The primer set and the kit adopt one-time PCR reaction. TGEV, PEDV, SADS-CoV and PDCoV can be identified rapidly. The specificity is good, the sensitivity is high, the repeatability and stability are good, and the primer set is suitable for differential diagnosis of TGEV, PEDV, SADS-CoV and PDCoV. The multi-PCR kit is quite suitable for pathogen diagnosis, pathogen purification detection and epidemiological investigation of epidemic diseases such as a laboratory, an entry and exit site and a farm.

Description

technical field [0001] The invention relates to the technical field of biotechnology, in particular to a multiplex RT-PCR detection primer set for TGEV, PEDV, SADS-CoV and PDCoV. Background technique [0002] Porcine diarrheal disease is a major cause of economic loss in free-range or large-scale pig farms, especially an important cause of death and stunting in suckling piglets. Since the occurrence of porcine diarrheal disease, it has been one of the problems that have plagued the breeding industry all over the world. In recent years, with the transformation of feeding methods to more large-scale and intensive feeding methods, the etiology of porcine diarrhea has become more and more complicated. Among all the factors causing diarrhea in pigs, viral diarrhea is the most important and serious factor, and the pathogen of viral diarrhea is the most harmful to piglets. The pathogens associated with viral diarrhea in piglets are mainly coronaviruses, including TGEV, PEDV, PDCo...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686C12Q2600/16C12Q2537/143C12Q2521/107Y02A50/30
Inventor 贺东生李锦辉李根
Owner SOUTH CHINA AGRI UNIV
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