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Duck viral hepatitis type 1 RT-LAMP detection kit and detection method thereof

A technology of RT-LAMP, duck viral hepatitis, applied in the directions of microorganism-based methods, biochemical equipment and methods, microbial determination/inspection, etc., can solve the disadvantages of rapid, accurate diagnosis, low specificity and sensitivity, detection Long time and other problems, to achieve the effect of being easy to popularize and apply on a large scale, ensuring high specificity, and shortening detection time

Inactive Publication Date: 2013-08-14
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there are many methods for detecting type 1 duck hepatitis antigen, such as virus neutralization test, immunoelectron microscopy and Dot-ELISA, and there are also many immune blood methods, such as ELISA, monoclonal antibody-PAP method, etc., but the above methods require The detection time is long, the specificity and sensitivity are low, and special equipment must be equipped, the operation is complicated, and non-specificity often occurs, which is not conducive to rapid and accurate diagnosis, so it has not been widely promoted and applied

Method used

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  • Duck viral hepatitis type 1 RT-LAMP detection kit and detection method thereof
  • Duck viral hepatitis type 1 RT-LAMP detection kit and detection method thereof
  • Duck viral hepatitis type 1 RT-LAMP detection kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: RT-LAMP detection of type 1 duck viral hepatitis

[0030] Prepare duck viral hepatitis RT-LAMP detection reaction solution according to the following composition:

[0031] (1) Reaction solution A: Contains 10× reaction buffer, Bst DNA polymerase, dNTP (2.5mM each), Betaine solution (5M), AMV, RNase Inhibitor, 0.1%TritonX-100, Mg 2+ , 2 pairs of primers whose sequences are:

[0032] F3: 3' GCTATGACATCAACCAATGT 5' (SEQ ID NO. 1)

[0033] B3: 3' TCTATCTCCATAGGGGCTAG 5' (SEQ ID NO.2)

[0034] FIP: 3'TAGTTTTGAGATATCGCGCACCTC--TGTCGTTAGTTATGGGGATGA 5' (SEQ ID NO. 3)

[0035] BIP: 3'TCGCACTATTTCAAGCTTTCTTTG--GTTATGTCACTTTCTTTGTCACTAG5' (SEQ ID NO. 4)

[0036] (2) Reaction solution B: 10000×SYBR green I

[0037] The system composition of 23 μL per tube of reaction solution A is as follows:

[0038]

[0039] (3) Preparation of RNA template for type 1 duck viral hepatitis

[0040] Type 1 duck viral hepatitis FC 64 The strain was inoculated into 9-day-ol...

Embodiment 2

[0047] Example 2: Sensitivity of RT-LAMP to detect type 1 duck hepatitis

[0048] (1) Preparation of duck hepatitis virus template

[0049] Type 1 duck viral hepatitis FC 64 The strain was inoculated with 9-day-old SPF chicken embryos, and the allantoic fluid was collected after the chicken embryos died. The viral RNA was extracted according to the instructions and literature of TRIzol Reagent (Invitrogen), and the quantification was 1.45 μg / μL, which was used as an RT-LAMP template. At the same time, 5 μL of RNA was reverse-transcribed into 20 μL of cDNA, which was diluted 10 times and used as a template for RT-PCR.

[0050] (2) Comparison of detection sensitivity between RT-LAMP and RT-PCR for type 1 duck hepatitis virus

[0051] In the reaction solution A, add 2 μL of duck hepatitis virus RNA of different concentrations prepared in the above-mentioned Example 1 as a template, mix well, bathe in water at 63°C for 40 minutes, inactivate in water bath at 80°C for 10 minute...

Embodiment 3

[0057] Example 3: Rapid RT-LAMP Detection Kit for Type 1 Duck Viral Hepatitis Virus

[0058] A type 1 duck hepatitis virus rapid detection kit comprises the following reagents:

[0059] Fluorescent dye SYBR green I, 10× reaction buffer, Bst DNA polymerase, dNTP (2.5mM each), Betaine solution (5M), AMV, RNase inhibitor, Mg 2+ , 0.1%TritonX-100, 2 pairs of primers, the sequence of which is:

[0060] F3: 3' GCTATGACATCAACCAATGT 5' (SEQ ID NO. 1)

[0061] B3: 3' TCTATCTCCATAGGGGCTAG 5' (SEQ ID NO.2)

[0062] FIP: 3'TAGTTTTGAGATATCGCGCACCTC-TGTCGTTAGTTATGGGGATGA 5' (SEQ ID NO. 3)

[0063] BIP: 3' TCGCACTATTTCAAGCTTTCTTTG-GTTATGTCACTTTCTTTGTCACTAG 5' (SEQ ID NO. 4)

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Abstract

The invention belongs to the technical field of biological detection and specifically relates to a duck viral hepatitis type 1 RT-LAMP detection kit and a detection method thereof. The invention discloses the duck viral hepatitis type 1 RT-LAMP detection kit and is characterized in that the kit contains two pairs of primers, the nucleotide sequence of which is as shown in SEQIDNO.1-4. The detection kit and its detection method provided by the invention have advantages of high speed, high sensitivity, high specificity, low cost and simple operation, can make up for the deficiency of present duck viral hepatitis detection methods, can satisfy the detection requirement of the disease at present, are easy for popularization and application at large scale, can be used to decrease the prevalence of the disease in ducks and other animals, and have a wide market prospect and great economic benefits.

Description

Technical field [0001] The present invention is a biological test technology field, which involves a type 1 duck virus hepatitis RT-LMP detection kit and its detection method. Background technique [0002] Duck Viral Hepatitis (DHV) is a virus disease that spreads rapidly and highly derived chucky ducks by three different internal types, which are type 1, type 2 and 3 duck hepatitis virus, of which Type 1 duck hepatitis is the most popular, and my country's main popular plants are also type 1 duck hepatitis.The virus mainly invasion of ducks within one week of age. The main pathological changes are liver enlargement, bleeding, etc., high incidence, high mortality rate, and ducks that are resistant to ducks.At present, one of the main infectious diseases in poultry breeding. [0003] At present, there are many methods for detecting type 1 duck hepatitis antigen, such as virus neutrality and testing, immune electromoscope and DOT-ELISA. There are also many immune blood methods, suc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 丁铲宋翠萍于圣青韩先干胡青海仇旭升谭磊
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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