Primers for loop-mediated isothermal amplification of avian reoviruses, detection kit of avian reoviruses and detection method

An avian reovirus, ring-mediated isothermal technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., to achieve the effects of high specificity and sensitivity, convenient observation and simple operation

Inactive Publication Date: 2012-07-18
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problems of low sensitivity and easy pollution in the detection of the above LAMP method, the present invention provides a ring-mediated isothermal amplification poultry call with high detection sensitivity Enterovirus primers

Method used

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  • Primers for loop-mediated isothermal amplification of avian reoviruses, detection kit of avian reoviruses and detection method
  • Primers for loop-mediated isothermal amplification of avian reoviruses, detection kit of avian reoviruses and detection method
  • Primers for loop-mediated isothermal amplification of avian reoviruses, detection kit of avian reoviruses and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1, the preparation of avian reovirus detection kit

[0063] 1. Synthesis of primers

[0064] Artificially synthesize the following 3 pairs of primers, the outer primer pair:

[0065] Upstream primer F3: 5'- ATGCTGCGTATGCCTCCC-3',

[0066] Downstream primer B3: 5'-CCAGCTCACGATGGAAGAC-3',

[0067] Inner primer pair:

[0068] Upstream primer FIP:

[0069] 5'-ACGTAGCTTGCAAATCACCACCTTTTTGTGTAACGGTGCGACTGCT-3',

[0070] Downstream primer BIP:

[0071] 5'- CATAATTGCATATTGGCCTTATCTTTTTTACGTGCAGCGTCCGCCTT-3',

[0072] Circular primer pair:

[0073] Upstream primer LF: 5'- GCCTGACAATGAACGTTACC-3',

[0074] Downstream primer LB: 5'- AGCGGCGGGTGGTGGTTTC-3',

[0075] 2. Preparation of LAMP reaction solution

[0076] Each 24μL LAMP reaction solution contains the following components: 20mM Tris-HCl, 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 4-16mM MgSO 4 , 0.1% Tween20, 0.05 mM Calcein, 0.6mM MnCl 2 , 0.2U AMV reverse transcriptase, 0.8M betaine, 1.4mM deoxynuc...

Embodiment 2

[0082] Get two chickens, one is diagnosed as a sick chicken infected with avian reovirus, and one is healthy. Two chickens were sampled separately with cloacal swabs. The detection kit prepared in Example 1 was used to detect the samples.

[0083] 1. Extraction of RNA

[0084] Collect the cloacal cotton swabs separately, rinse the cotton swabs repeatedly in a centrifuge tube with 0.2mol / L pH7.4 phosphate buffer solution, take out the cotton swabs, centrifuge at 500Orpm for 1min, and take the supernatant to obtain the test solution of 2 chickens.

[0085] The operation of extracting RNA is an existing technology, and only one of the specific operations is listed as follows, but it is not limited to this kind of operation:

[0086] Add 250 μL of detection solution and 750 μL TRIzol Reagent to a centrifuge tube, shake vigorously, and ice-bath for 5 minutes; add 200 μL of chloroform, and ice-bath for 5 minutes; centrifuge at 12,000 rpm for 10 minutes at 4°C, take the aqueous...

Embodiment 3

[0094] Get two chickens, one is diagnosed as a sick chicken infected with avian reovirus, and one is healthy. Two chickens were sampled separately with cloacal swabs. The detection kit prepared in Example 1 was used to detect the samples.

[0095] 1. Extraction of RNA

[0096] Same as Step 1 of Example 2.

[0097] 2. Loop-mediated isothermal amplification

[0098] Add 1 μL to each of the three reaction tubes containing 24 μL of the LAMP reaction solution to obtain the RNA sample of the diseased chicken in step 1, as the detection group 1; add 1 μL to each of the three reaction tubes containing 24 μL of the LAMP reaction solution. Get the RNA samples of healthy chickens as the detection group 2; add 1 μL positive control to each of the three reaction tubes containing 24 μL of LAMP reaction solution, as the positive control group;

[0099] 1 μL of sterilized double-distilled water was added to each reaction tube as a negative control group.

[0100] At the same time, the r...

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Abstract

The invention relates to the technical field of avian reovirus detection, in particular to primers for loop-mediated isothermal amplification of the avian reoviruses, comprising three pairs of specific primers, and the sequences of the primers are shown in a sequence list. A detection kit of the avian reoviruses comprises loop-mediated isothermal amplification reaction liquid which contains the three pairs of specific primers. A detection method of the avian reoviruses is characterized in that the detection kit is used for carrying out the loop-mediated isothermal amplification reaction on anRNA sample, a positive control group and a negative control group are set, and the conditions of the loop-mediated isothermal amplification reaction are that the temperature is 59-65 DEG C, reaction time is 30-75 minutes. The detection kit of the avian reoviruses has high specificity and sensitivity, and by using the detection kit, 100 copies of RNA can be detected, the operation is simple, and observation is convenient.

Description

[0001] technical field [0002] The invention relates to the technical field of avian reovirus detection, in particular to primers for ring-mediated isothermal amplification of avian reovirus, a detection kit for avian reovirus, and the avian reovirus detection method. Background technique [0003] Avian reoviruses (ARV) belong to the Orthoreovirus genus of the Reoviridae family and mainly infect chickens, turkeys, ducks, geese and other birds. Avian reovirus mainly causes arthritis and tenosynovitis in poultry, but also growth retardation, pericarditis, myocarditis, hepatitis, osteoporosis, and acute and chronic respiratory diseases. In addition, the mixed infection of avian reovirus and other immunosuppressive diseases is common in chicken flocks, resulting in slow growth of chickens, low feed return rate, affecting immune effect, and causing huge economic losses to the poultry industry. [0004] At present, there are many diagnostic methods for avian reovirus, mainly ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 张琳张秀美胡北侠杨少华颜世敢许传田
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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