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RT-LAMP (reverse transcription loop-mediated isothermal amplification) primer set and kit for detecting porcine epidemic diarrhea virus and application thereof

A technology of RT-LAMP and porcine epidemic diarrhea, which is applied in the detection field of porcine epidemic diarrhea virus, can solve the problems of being unsuitable for grass-roots or on-site rapid detection, requiring expensive instruments and equipment, and long detection cycle, etc., to achieve a wide range of objects, The equipment and operation are simple and the applicability is strong

Inactive Publication Date: 2015-12-23
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These methods have played an important role in virus detection, but they generally have disadvantages such as cumbersome operation, long detection cycle, and need for expensive instruments and equipment, and are not suitable for rapid detection at the grassroots level or on-site

Method used

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  • RT-LAMP (reverse transcription loop-mediated isothermal amplification) primer set and kit for detecting porcine epidemic diarrhea virus and application thereof
  • RT-LAMP (reverse transcription loop-mediated isothermal amplification) primer set and kit for detecting porcine epidemic diarrhea virus and application thereof
  • RT-LAMP (reverse transcription loop-mediated isothermal amplification) primer set and kit for detecting porcine epidemic diarrhea virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 detects the best embodiment of the kit of porcine epidemic diarrhea virus with RT-LAMP method

[0043] The kit of this embodiment includes the following RT-LAMP primer sets, see Table 1. The kits are divided into A box and B box, and A box is an RNA extraction kit. The composition and storage conditions of the kit are shown in Table 2. Box B is a porcine epidemic diarrhea virus reverse transcription loop-mediated isothermal amplification (RT-LAMP) kit. The kit composition and storage conditions are shown in Table 3.

[0044] Among them, the RT-LAMP reaction solution is composed of 50uL 10×BstDNABuffer, 50uL MgCl2 solution with a concentration of 25mM, 60uL dNTPs solution with a concentration of 2.5mM, and 120uL nuclease-free water.

[0045] The reverse transcriptase consists of 20 uL of AMV reverse transcriptase at a concentration of 200 U / μL and 10 uL of an RNase inhibitor at a concentration of 50 U / μL.

[0046] The fluorescent chromogenic reagent contai...

Embodiment 2

[0053] Embodiment 2 non-diagnostic purpose detects the detection method of porcine epidemic diarrhea virus with RT-LAMP method

[0054] The detection method of this embodiment uses the kit of Embodiment 1 for detection. The detection method of this embodiment comprises the following steps:

[0055] 1. Viral RNA extraction Porcine epidemic diarrhea virus RNA was extracted by one-step method of guanidine isothiocyanate-phenol-chloroform. The specific steps are as follows: (1) Denaturation. Add 750 μL TRIzolLS Reagent to 250 μL of the sample to be tested, the positive control and the negative control, shake vigorously for 2 minutes, and place at room temperature for 10 minutes. Add 250 μL of chloroform, shake vigorously for 15 s, leave at room temperature for 10 min, centrifuge at 12,000 g for 10 min at 4°C, and transfer the upper aqueous phase (about 500-600 μL) into a new centrifuge tube. Wherein, the samples to be tested can be porcine feces with diarrhea, intestinal tract ...

Embodiment 3

[0063] The specificity test of the RT-LAMP of embodiment 3 porcine epidemic diarrhea virus

[0064] According to the process of embodiment 2, porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus (PRRSV), porcine transmissible gastroenteritis virus (TGEV), rotavirus (RV) are respectively carried out RT-LAMP reaction, detect Specificity of the RT-LAMP method. The results are attached figure 1 As shown, the nucleic acid of porcine epidemic diarrhea virus amplified a unique "S" curve, while the nucleic acids of the other three porcine diseases did not amplify the "S" curve. The above results show that the RT-LAMP detection method of the present invention can specifically amplify porcine epidemic diarrhea virus without cross-reaction with other porcine disease virus nucleic acids, and the RT-LAMP method of the present invention has good specificity.

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Abstract

The invention discloses an RT-LAMP (reverse transcription loop-mediated isothermal amplification) primer set and kit for detecting porcine epidemic diarrhea virus and application thereof. The primer set is composed of a pair of outer primers, a pair of inner primers and a pair of loop primers. Sequences of the paired outer primers are shown in SEQ ID No.1 and SEQ ID No.2. Sequences of the paired inner primers are shown in SEQ ID No.3 and SEQ ID No.4. Sequences of the paired loop primers are shown in SEQ ID No.5 and SEQ ID No.6. The primer set is effective in detecting the porcine epidemic diarrhea virus. The kit comprises the afore-mentioned primers and is applicable to detecting the porcine epidemic diarrhea virus. The invention further provides a method of detecting the porcine epidemic diarrhea virus with the kit; detection results of the method may be visually detected according to changes in the color of reaction liquid, quickly and accurately. The primer set, the kit and the method are highly specific, highly sensitive and quick and accurate in detecting the porcine epidemic diarrhea virus and are particularly applicable to quick field detection.

Description

technical field [0001] The invention relates to the technical field of detection of porcine epidemic diarrhea virus. Background technique [0002] Porcine epidemic diarrhea (porcineepidemicdiarrheavirus, PED) is caused by porcine epidemic diarrhea virus (porcineepidemicdiarrheavirus, PEDV), a highly contagious disease of pigs with diarrhea, vomiting and dehydration as the main clinical features. It first appeared in the UK in 1971, and the disease occurred successively in my country in the early 1980s. At present, the disease has been reported in many major pig-raising countries in the world. [0003] PEDV detection methods mainly include: virus isolation, RT-PCR, real-time fluorescence quantitative PCR. These methods have played an important role in virus detection, but they generally have disadvantages such as cumbersome operation, long detection cycle, and expensive equipment, and are not suitable for rapid detection at the grassroots level or on-site. [0004] The rev...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2600/166C12Q2531/119C12Q2521/107C12Q2547/101C12Q2545/101
Inventor 袁万哲孙继国宋勤叶马增军李丽敏郭红斌李亚楠
Owner HEBEI AGRICULTURAL UNIV.
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