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LAMP (Loop-medicated isothermal amplification) detection kit for metapneumovirus

The technology of a poultry pneumonia virus and kit is applied in the direction of microbial determination/testing, recombinant DNA technology, and microbial-based methods, which can solve the problems of high cost, time-consuming, unsuitable for grass-roots promotion and use, and achieve low cost and high detection efficiency. Fast, easy-to-use effects

Active Publication Date: 2013-02-27
GUANGXI VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional chicken embryo isolation method of virus is accurate and simple, but it takes too long, usually takes two days to a week; IFA and ELISA are fast but require special reagents; RT-PCR, RRT-PCR, Gene chips detection technology is fast and sensitive , but all require special instruments and technical personnel, and the cost is relatively high, so it is not suitable for promotion and use at the grassroots level

Method used

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  • LAMP (Loop-medicated isothermal amplification) detection kit for metapneumovirus
  • LAMP (Loop-medicated isothermal amplification) detection kit for metapneumovirus
  • LAMP (Loop-medicated isothermal amplification) detection kit for metapneumovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, LAMP primer design and kit of avian pneumonia virus

[0038] 1. Primer design

[0039] According to the conserved region of the F gene of avian pneumonia virus (Avian pneumonia virus, APV) (Genbank number: AF187154, as shown in sequence number 7), the following LAMP primers were designed and synthesized (the directions are all from the 5'-3' end):

[0040] Internal primer 1: AGCAGGACAATGAGGACTATCACTA-GTCAACAAGATATTGGATAGCAC (as shown in sequence listing sequence 1, the sequence before the horizontal line is complementary to the 1515-1491th position of the sequence listing sequence 7, and the sequence after the horizontal line is the same as the 1440-1463rd position of the sequence listing sequence 7) ;

[0041]Internal primer 2: AGTTGGTGTGGGTGTCTTCT-CCATTCATTTCCATTGGGAA (as shown in sequence listing sequence 2, the sequence before the horizontal line is the same as the 1528-1547th position of the sequence listing sequence 7, and the sequence after the hor...

Embodiment 2

[0049] Embodiment 2, the specific detection of LAMP kit

[0050] 1. Obtaining the template

[0051] 1. Viral RNA extraction and cDNA acquisition

[0052] 1) Viral RNA extraction

[0053] Using Trizol reagent extraction (Invitrogen Company, product number 15596-026) according to the instruction manual, RNA was extracted from chicken embryo allantoic fluid infected with the following pathogens respectively (the samples obtained by extracting negative chicken embryo allantoic fluid were used as negative control samples ): Avian pneumonia virus strain MN2a (confirmed to be avian pneumonia virus by sequencing comparison), chicken Marek's virus strain MDV (Nanjing Merial Animal Health Products Co., Ltd.), chicken infectious bronchitis virus strain Mass41, avian reo Orphan virus strain S1133, chicken Newcastle disease virus strain Lasota and avian encephalomyelitis virus strain VanRoekel.

[0054] 2) Acquisition of cDNA

[0055] The RNA samples obtained in step 1) were reverse-tr...

Embodiment 3

[0070] Embodiment 3, sensitivity detection of LAMP kit

[0071] 1. Preparation of RNA samples with different concentrations

[0072] The RNA of the avian pneumonia virus MN2a strain obtained in Step 1 of Example 2 was quantified and then diluted in 10-fold increments to 100ng / μL, 10ng / μL, 1ng / μL, 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL, 10fg / μL, 1fg / μL.

[0073] 2. Acquisition of cDNA

[0074] The different concentrations of RNA samples obtained in Step 1 were reverse transcribed according to the method of 1-2) in Step 1 of Example 2 to obtain cDNA.

[0075] 3. LAMP amplification and results

[0076] Take the cDNA sample in step 2 and carry out LAMP amplification according to the reaction system and reaction procedure in step 2 of Example 2 respectively, and the observation results under visible light are as follows: figure 2 As shown, 1 in the figure is 1ng / μL; 2 is 100pg / μL; 3 is 10pg / μL; 4 is 1pg / μL; 5 is 100fg / μL; 6 is 10fg / μL; 7 is 1fg / μL; -5 are all positive, and 6-7 ...

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Abstract

The invention discloses an LAMP (loop-medicated isothermal amplification) detection kit for metapneumovirus. The kit internally comprises a primer set formed by six primers designed aiming at eight regions of a metapneumovirus F gene conserved region, wherein the nucleotide sequence of the primer set is as shown in a sequence table from sequence 1 to sequence 6. The detection kit disclosed by the invention can be used for specifically detecting the metapneumovirus, wherein the minimum detection limit of the detection kit is 100fg / muL, and the sensitivity of the detection kit is 100 times as high as that of the normal RT-PCR (reverse transcription-polymerase chain reaction). The LAMP detection kit is quick to detect, low in cost, free from expensive apparatuses, simpler in operation method, and suitable for the quick detection in the clinic.

Description

technical field [0001] The invention relates to a LAMP detection kit for avian pneumonia virus. Background technique [0002] Avian pneumonia virus (APV) is a member of the genus Avian Metapneumovirus and belongs to the family Avian Paramyxoviridae. The virus mainly harms turkeys and chickens of different breeds, including breeders, commercial broilers and eggs. The age of onset is generally 4-7 weeks old, and the peak of the disease is 5-6 weeks old. It mainly causes upper respiratory symptoms, such as sneezing. , conjunctival flushing, lacrimal gland swelling, subcutaneous edema on the head, commonly known as swollen head; infection of laying hens mainly causes egg production to drop by 2%-40%, and hatching rate decreases; as the disease progresses, it also shows neurological symptoms; The morbidity rate caused by the disease is between 1% and 90%, and the mortality rate of chickens is 1% to 20%. During the morbidity, the infection of other diseases will cause greater dea...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 谢芝勋谢志勤刘加波庞耀珊邓显文谢丽基范晴罗思思
Owner GUANGXI VETERINARY RES INST
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