Nested RT-PCR primer set and reagent kit for detecting atypical porcine pestivirus virus, and application of nested RT-PCR primer set and reagent kit
A detection kit and a typical technology, applied in the field of nested RT-PCR primer sets for the detection of porcine atypical fever virus, can solve the problems of small tissue sample requirements, inability to diagnose at one time, large damage, etc., and achieve high Sensitivity and specificity, improved detection rate, high sensitivity effect
Inactive Publication Date: 2019-08-06
SOUTH CHINA AGRI UNIV
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Problems solved by technology
[0005] Conventional virus detection techniques such as pathological slides and immunohistochemistry have disadvantages such as long cycle time, large damage, and inability to make a one-time diagnosis, while polymerase chain reaction (PCR) technology is fast, sensitive, and requires a small amount of tissue samples. Especially suitable for pathogen monitoring
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Embodiment 1
[0035] 1. Design and synthesis of nested RT-PCR primers for detection of atypical swine fever virus (APPV)
[0036] According to the reference and comparison of the conserved sequence of the APPV gene published in GenBank, the MegAlign software was used for sequence alignment, and the NS5B region of the conserved gene fragment located in the whole gene sequence was selected as the primer design region, and the height of the conserved nucleotide sequence was determined. The conserved fragment was used as the amplified region, and three sets of nested RT-PCR primer sets for detecting APPV were designed by using the primer-assisted design software primer5 according to the determined conserved nucleotide sequence: including the forward primer APPV-X-F1 and the reverse primer APPV - the outer primer composed of X-R1 and the inner primer composed of forward primer APPV-X-F2 and reverse primer APPV-X-R2 (X is 1, 2 or 3, representing the first group, the second group, the Three sets o...
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The invention provides a nested RT-PCR primer, method and reagent kit for detecting an atypical porcine pestivirus virus. According to the reagent kit provided by the invention, a plurality of sets ofnested PCR primers are designed according to the highly-conserved specific sequence of the atypical porcine pestivirus virus, through primer screening and reaction system optimization, a nested PCR detection reagent kit which is high in detection sensitivity and good in specificity is researched and developed. According to the detection reagent kit provided by the invention, only a common PCR instrument is needed, the detection sensitivity equivalent to that through real-time fluorescent quantitation PCR can be obtained, the micro latent infection of the atypical porcine pestivirus virus canbe monitored, and early-stage early warning of the atypical porcine pestivirus virus can be realized; and besides, the detection cost is low, requirements for detecting instruments and equipment and the operating level of staff are low, and application and promotion of the detection reagent kit to breeding and producing frontlines are facilitated.
Description
technical field [0001] The invention belongs to the technical field of animal epidemic detection, and in particular relates to a nested RT-PCR primer set, a kit and an application for detecting porcine atypical plague virus. Background technique [0002] Atypical swine fever (APP) is congenital tremor in piglets (commonly known as "shivering disease") caused by atypical porcine pestivirus virus (APPV). , Severe can lead to tremors all over the body, difficulty in breastfeeding. [0003] Porcine atypical pestivirus (APPV) is a single-stranded positive-sense RNA virus of the genus Pestivirus in the family Flaviviridae. The genome is approximately 12KB, including 5' and 3' non-coding regions and a polyprotein reading frame (ORF). APPV is a member of the Flaviviridae and Pestivirus genus, which also includes bovine viral diarrhea virus types 1 and 2 (bvdv-1 and bvdv-2), swine fever virus (CSFV) and border disease virus (BDV), among others Virus. [0004] Initially, Arruda et...
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Login to View More IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/686C12Q1/701
Inventor 贺东生韦柳明苏丹萍谢永生李锦辉梁伟放司广斌
Owner SOUTH CHINA AGRI UNIV