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A molecular kit and its application for rapid identification of three types of piglet viral diarrhea

A porcine epidemic diarrhea and kit technology, which is applied in the directions of microorganism-based methods, microorganism determination/inspection, microorganisms, etc., can solve the problems of long time consumption, and achieve the effects of short time consumption, strong specificity and high sensitivity

Active Publication Date: 2018-02-23
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Traditional methods for detecting these three diseases, such as virus isolation and identification and serological examination, often take too long

Method used

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  • A molecular kit and its application for rapid identification of three types of piglet viral diarrhea
  • A molecular kit and its application for rapid identification of three types of piglet viral diarrhea
  • A molecular kit and its application for rapid identification of three types of piglet viral diarrhea

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The preparation of embodiment 1 kit of the present invention

[0038] 1. Materials and instruments

[0039] The same experimental materials and instruments as mentioned above.

[0040] 2. Experimental method

[0041] 2.1PCR primer design

[0042] According to the N gene of porcine transmissible gastroenteritis virus TGEV (GenBank accession number: DQ443743), the M gene of porcine epidemic diarrhea disease PEDV (GenBank accession number: AY974335), the VP7 gene of porcine rotavirus PoRV (GenBank accession number: X04613.1), according to the principle of multiplex PCR primer design, each designed and synthesized a pair of primers; and designed three pairs of primers for constructing positive plasmids, and the primers were synthesized by Shanghai Sangong Biotechnology Co., Ltd. After the primer is synthesized, it is in the form of a dry powder that cannot be seen clearly by the naked eye. It needs to be centrifuged and dissolved in sterilized ultrapure water. The primer...

Embodiment 2

[0125] Embodiment 2 specificity experiment

[0126] 1. Test method

[0127] Viral RNA or DNA such as TGEV, PEDV, PoRV, CSFV, PRRSV, JEV, and PRV were extracted respectively, and amplified using the three pairs of primers in Example 1, and DEPC-treated water was used as a blank control. To verify the specificity of the gene chip and kit of the present invention.

[0128] Reaction conditions: 95°C for 2min, 95°C for 30s, annealing temperature at 58.8°C for 30s, 72°C for 1min, 30 cycles, 72°C for 5min, storage at 16°C.

[0129] The reaction system is as follows:

[0130]

[0131] 2. Results

[0132] Experimental results such as Figure 13 Shown, adopt the method of the present invention to detect, PEDV, TGEV, PoRV detection result is positive, and the detection result of other virus is all negative.

[0133] The method of the present invention can only detect 3 kinds of viruses of the present invention, but not other viruses, which shows that the specificity of the kit of...

Embodiment 4

[0134] Embodiment 4 sensitivity test

[0135] 1. Test method

[0136] Adjust the RNA copy numbers of TGEV, PEDV, and PoRV to 1.5×10 8 Copies / μL, the three templates were mixed and diluted 10-fold, and reverse-transcribed into cDNA according to the instructions of the reverse transcription kit (Bao Biology) and amplified with the three pairs of primers in Example 1, while DEPC-treated water was used as a blank control.

[0137] Reaction conditions: 95°C for 2min, 95°C for 30s, annealing temperature at 58.8°C for 30s, 72°C for 1min, 30 cycles, 72°C for 5min, storage at 16°C.

[0138] The reaction system is as follows:

[0139]

[0140] 2. Results

[0141] The result is as Figure 14 As shown, the detection of 10-fold serially diluted positive RNA templates shows that the target bands can be seen in lanes 1-4, and 10 4 The prepared positive RNA template was diluted twice, that is, the concentration was 1.5×10 4 copies / µL of sample.

[0142] Experimental result shows, a...

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Abstract

The invention discloses a kit for detecting pig epidemic diarrhea viruses, pig transmissible gastroenteritis viruses and pig rotaviruses. The kit comprises primer pairs shown by SEQ ID NO:1-2, SEQ ID NO:3-4 and SEQ ID NO:5-6 for respectively performing specific amplification on the pig epidemic diarrhea viruses, the pig transmissible gastroenteritis viruses and the pig rotaviruses. The invention also discloses applications of the primer pairs shown by the SEQ ID NO:1-2, the SEQ ID NO:3-4 and the SEQ ID NO:5-6 in preparation of reagents for detecting the pig epidemic diarrhea viruses, the pig transmissible gastroenteritis viruses and the pig rotaviruses. The detection kit disclosed by the invention can be used for accurately and effectively detecting the pig epidemic diarrhea viruses, the pig transmissible gastroenteritis viruses and the pig rotaviruses, and is strong in specificity, high in sensitivity, short in time consumption, rapid in detection and good in application prospect.

Description

technical field [0001] The invention relates to a kit for detecting porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine rotavirus. Background technique [0002] With the increasing scale of centralized pig farming, the diarrheal diseases of pigs are becoming more and more serious. The research on the pathogens causing diarrhea in piglets has confirmed that there are and are still increasing Escherichia coli, Salmonella, Clostridium welchii, porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine rotavirus, mild swine fever, swine dysentery , Coccidia etc. Piglet viral diarrhea diseases caused by porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, and porcine rotavirus are increasing day by day. These three types of diseases can cause high morbidity and mortality in piglets, and in some areas The phenomenon of mixed infection and secondary infection has appeared, which has caused gr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
CPCC12Q1/701C12Q2600/16
Inventor 黄小波曹三杰文心田朱书权邓丽滑翔文翼平伍锐马晓平张雨迪卿盈
Owner SICHUAN AGRI UNIV
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