LAMP visual quick detection kit for porcine circovirus type 2

A porcine circovirus and detection kit technology, applied in the field of agricultural biology, can solve the problems of simple operation and complex LAMP method principle, and achieve the effects of simple operation, convenient result reading and high sensitivity

Inactive Publication Date: 2017-10-31
SHANGHAI ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The principle of the LAMP method is complicated, but the actual operation is simple

Method used

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  • LAMP visual quick detection kit for porcine circovirus type 2
  • LAMP visual quick detection kit for porcine circovirus type 2
  • LAMP visual quick detection kit for porcine circovirus type 2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Design of LAMP primers for detection of porcine circovirus type 2

[0036] The present invention designs 4 specific primers based on 6 different regions (F3c, F2c and Flc regions at the 3'end and Bl, B2 and B3 regions at the 5'end) on the porcine circovirus type 2 (PCV2) ORF2 gene sequence in GenBank , And design a pair of loop primers: LF and LB according to the target gene. The specific sequence is shown in Table 1.

[0037] Table 1

[0038]

[0039] The FIP primer of the present invention is an upstream internal primer (Forward Inner Primer), which is composed of an F2 region and an F1c region. The F2 region is complementary to the F2c region at the 3'end of the target gene, and the F1c region has the same sequence as the Flc region at the 5'end of the target gene. The F3 primer is an upstream outer primer (Forward Outer Primer), which consists of the F3 region and is complementary to the F3c region of the target gene. The BIP primer is a downstream inner primer...

Embodiment 2

[0043] Example 2 The LAMP rapid detection method of porcine circovirus type 2 includes the following steps:

[0044] 1) Extract the total DNA of the sample to be tested as a reaction template.

[0045] 2) Prepare LAMP detection system: the total reaction volume is 25μL, including: ddH 2 O 4.5μL, 1×dNTP 3.0μL, 10×buffer 2.5μL, primer FIP 2μL, primer BIP 2μL, primer F30.5μL, primer B3 0.5μL, primer LF 2μL, primer LB 2μL, betaine 4μL, Bst enzyme 1μL, The specific preparation of the detection system is as follows:

[0046] Add substances other than Bst enzyme into a 1.5mL centrifuge tube, vortex to mix, and instantaneously separate; heat the centrifuged mixture in a 95°C water bath for 5 minutes, and then place it on ice for 5 minutes. Add 1μLBst enzyme to the centrifuge tube, use a vortex mixer to stir the various substances in the centrifuge tube evenly, and then place it on the centrifuge for instant centrifugation to obtain the LAMP detection system.

[0047] 3) Take 1 μL of the tota...

Embodiment 3

[0049] Example 3 Assembly of LAMP Visualization Rapid Detection Kit for Porcine Circovirus Type 2

[0050] 1. Packing specification: 200 times

[0051] 2. The composition of the kit is shown in Table 2:

[0052] Table 2

[0053]

[0054] *: Contains Buffer, dNTP Mix and Betaine.

[0055] 3. Transportation and storage methods:

[0056] Transport at low temperature. For short-term use, keep it at 4℃ and avoid light. For long-term storage, please store it in a refrigerator at -20℃ or -80℃.

[0057] 4. Precautions for use:

[0058] (1) All items, all materials that have been in contact with viral materials, need to be treated reasonably, in order to prevent them from contaminating and contaminating the laboratory and causing adverse effects on personnel.

[0059] (2) Divide the entire experiment into regions. Backflow of equipment and reagents is strictly prohibited.

[0060] (3) The developer is low-toxic and should be stored at room temperature and protected from light. Wear gloves during ope...

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Abstract

The invention discloses an LAMP visual quick detection kit for porcine circovirus type 2, and discloses an LAMP primer for detecting the porcine circovirus type 2 and a detection method thereof. Three pairs of primers are designed and synthesized according to the ORF2 gene sequence of the porcine circovirus type 2 (PCV2), an LAMP diagnosing method of the PCV 2 is established by optimizing the LAMP condition, a simple and quick method is provided to field quick diagnosis of the porcine circovirus type 2, and the LAMP visual quick detection kit for the PCV2 is prepared according to the method. The kit is simple in operation, and can directly determine the result of color changes to realize visualization, has easily determined reaction results, and can be easily popularized in clinical application.

Description

Technical field [0001] The invention belongs to the field of agricultural biotechnology, and specifically relates to a LAMP visual rapid detection kit for porcine circovirus type 2, and more specifically to a LAMP primer, a detection method and a LAMP visual rapid detection reagent for detecting porcine circovirus type 2 box. Background technique [0002] Porcine circovirus type 2 (PCV2) belongs to the circovirus genus of the Circoviridae family. It is a single-stranded negative-strand circular DNA virus without an envelope. It is the main pathogen that causes post-weaning multiple system failure syndrome (PMWS) in piglets. At present, the disease is widespread worldwide, causing serious economic losses in pig production. In addition, PCV2 is also related to porcine infectious congenital tremor granulomatous enteritis dermatitis, nephrotic syndrome porcine respiratory syndrome porcine reproductive disorder exudative epidermatitis and necrotizing lymphadenitis and other diseases....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2563/107
Inventor 赵凯倪建平赵笑胡瑞丽吴洋洋李春华赵庆史斌赵桓震赵斌安潘磊范小瑞赵本进蒋风英张春玲
Owner SHANGHAI ACAD OF AGRI SCI
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