Duplex fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for Nipah virus and porcine reproductive and respiratory syndrome virus (PRRSV) as well as preparation method and application of duplex fluorescent RT-PCR detection reagent

A technology of RT-PCR and respiratory syndrome, applied in the direction of biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc.

Inactive Publication Date: 2015-07-01
ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Claims
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Problems solved by technology

At present, the fluorescent RT-PCR method and published patents for the detection of single Nipah virus and porcine reproductive and respiratory syndrome virus have been reported, and the dual fluorescent RT-PCR detection that can simultaneously detect Nipah virus and porcine reproductive and respiratory syndrome virus Reagents and their preparation methods and applications are rarely reported

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  • Duplex fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for Nipah virus and porcine reproductive and respiratory syndrome virus (PRRSV) as well as preparation method and application of duplex fluorescent RT-PCR detection reagent
  • Duplex fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for Nipah virus and porcine reproductive and respiratory syndrome virus (PRRSV) as well as preparation method and application of duplex fluorescent RT-PCR detection reagent
  • Duplex fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for Nipah virus and porcine reproductive and respiratory syndrome virus (PRRSV) as well as preparation method and application of duplex fluorescent RT-PCR detection reagent

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preparation example Construction

[0048] The double fluorescent RT-PCR detection reagent for Nipah virus and porcine reproductive and respiratory syndrome virus and its preparation method and application of the present invention comprise the following steps.

[0049] The first step is to prepare the positive control substance (RNA) of Nipah virus M gene, and the process includes:

[0050] (1) On the NCBI website, perform BLAST comparison on the M gene sequence of Nipah virus, and select a conserved sequence for the preparation of the M gene positive control, as shown in SEQ ID NO.4.

[0051] (2) Design and synthesize 4 primers based on the sequence of SEQ ID NO.4, the primer sequences are as follows:

[0052] NIPHA-M-F1:5'-CCTGGAACAACAGTTGTGAGATCAGCCGAGTAGCAGCTGTGTTGCAGCCTTCTGTTCC-3

[0053] NIPHA-M-R1:5'-GGAACAGAAGGCTGCAACACAGCTGCTACTCGGCTGATCTCACAACTGTTGTTCCAGG-3

[0054] NIPHA-M-F2:5'-GATGGACATCAATCCTTGGCTCAACAGATTGACCTGGAACAACAGTTGTGAGATC-3'

[0055] NIPHA-M-R2:5'-GAAGACATCATCATAGATCATGAACTCTCTTGGAACAGA...

Embodiment 1

[0103] Example 1 Preparation of Nipah virus M gene positive control

[0104] 1. Selection of reference gene sequence

[0105] BLAST was performed on the Nipah virus M gene sequence (GenBank accession number: AJ564621.1), and a conservative and suitable sequence for designing fluorescent RT-PCR primers and probes was selected as a reference sequence for preparing Nipah virus M gene positive control (RNA) , as shown in SEQ ID NO.1.

[0106] 2. Design and synthesis of amplification primers

[0107] Based on the above sequences, 4 PCR amplification primers were designed and synthesized, in which NIPHA-M-F1 and NIPHA-M-R1 sequences were completely complementary and used as templates, and NIPHA-M-F2 and NIPHA-M-R2 were respectively compatible with Partially overlapping with the NIPHA-M-R1 sequence, the synthesized sequence is as follows:

[0108] NIPHA-M-F1:5'-CCTGGAACAACAGTTGTGAGATCAGCCGAGTAGCAGCTGTGTTGCAGCCTTCTGTTCC-3

[0109] NIPHA-M-R1:5'-GGAACAGAAGGCTGCAACACAGCTGCTACTCGGCTG...

Embodiment 2

[0125] Example 2 Preparation of Porcine Reproductive and Respiratory Syndrome Virus NSP2 Gene Positive Control (RNA)

[0126] 1. Selection of reference gene sequence

[0127] BLAST was performed on the porcine reproductive and respiratory syndrome virus NSP2 gene sequence (GenBank accession number: JX087437), and a conservative and suitable sequence for designing fluorescent RT-PCR primers and probes was selected as a positive control for porcine reproductive and respiratory syndrome virus NSP2 gene (RNA) reference sequence, as shown in SEQ ID NO.1.

[0128] 2. Design and synthesis of amplification primers

[0129] Based on the above sequences, 4 PCR amplification primers were designed and synthesized, in which PRRSV-NSP2-F1 and PRRSV-NSP2-R1 sequences were completely complementary and used as templates, and PRRSV-NSP2-F2 and PRRSV-NSP2-R2 were respectively combined with PRRSV-NSP2-F1 and

[0130] The sequences of PRRSV-NSP2-R1 partially overlap, and the synthesized sequenc...

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Abstract

The invention discloses a duplex fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent for a Nipah virus and porcine reproductive and respiratory syndrome virus (PRRSV) as well as a preparation method and application of the duplex fluorescent RT-PCR detection reagent. Two sets of specific primers and Taqman probes which are respectively used for an M gene of the nipah virus and an NSP2 gene of the PRRSV as well as positive control are designed and synthesized; a duplex fluorescent RT-PCR detection system with the advantages of quickness, simplicity, convenience, high specificity and high sensitivity is established by using the two sets of primers and probes; the duplex fluorescent RT-PCR detection system can be used for simultaneously detecting nucleic acids of the Nipah virus and the PRRSV quickly, accurately, specifically, safely, simply and conveniently from a detected sample within 3 to 4 hours, and simultaneously detecting nucleic acids of trace Nipah virus and the PRRSV from a pig and a correlated sample of the pig.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a double fluorescent RT-PCR detection reagent for Nipah virus and porcine reproductive and respiratory syndrome virus, a preparation method and application thereof. Background technique [0002] Nipah virus (NiV) is a new virus identified in 1999 that seriously harms domestic pigs and humans. It can cause nervous system and respiratory diseases in humans and pigs. Porcine respiratory and reproductive syndrome is a highly contagious infectious disease of pigs caused by porcine respiratory and reproductive syndrome virus. Because Nipah disease and porcine respiratory and reproductive syndrome have similar clinical manifestations in pigs, it is not easy to distinguish them based on clinical symptoms alone. It can provide etiological basis for early detection and control of these two animal diseases, and can also prevent and control the outbreak and prevalence of human Nipah...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68C12Q1/70
CPCC12Q1/701
Inventor 王建华赵丹肖妍张俊哲赵祥平董志珍王玉玲陈小金王乃福陈本龙
Owner ANIMAL & PLANT & FOOD INSPECTION CENT OF TIANJIN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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