Fluorescent RT-PCR reagent kit for detecting Japanese B encephalitis virus

A Japanese encephalitis virus, RT-PCR technology, applied in the field of bioengineering, can solve the problems of unfavorable large-scale sample detection, long time for kits, complicated procedures, etc., and achieves the effects of easy result judgment, simple and fast method, and simple preparation.

Inactive Publication Date: 2015-07-22
SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Aiming at the above technical problems in the prior art, the invention provides a fluorescent RT-PCR kit for detecting Japanese encephalitis virus, said fluorescent RT-PCR kit for detecting Japanese encephalitis virus -The PCR ki...

Method used

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  • Fluorescent RT-PCR reagent kit for detecting Japanese B encephalitis virus
  • Fluorescent RT-PCR reagent kit for detecting Japanese B encephalitis virus
  • Fluorescent RT-PCR reagent kit for detecting Japanese B encephalitis virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] 1. Design and optimization of primers and probes

[0030] Sequence source: multiple strains of porcine Japanese encephalitis virus were searched through the NCBI gene bank, and a representative strain of the strain was downloaded from the gene bank. The accession number of the representative strain is JX040321.1. The sequences in the prM region of the porcine Japanese encephalitis virus genome were used as reference sequences for the design of primers and probe sequences.

[0031] prM represents the sequence reference sequence shown in SEQ ID NO: 1, with a full length of 501 bp.

[0032] By primer design software (Primer Express 3.0.1), the following sequence was designed:

[0033] Upstream primer J1: 5'-ACCAAGAAGTCTACGTCCAATATGG-3' (as shown in SEQ ID NO: 2);

[0034] Downstream primer J2: 5'-TTGGACCGACACGGATCTC-3' (as shown in SEQ ID NO: 3);

[0035] TaqMan probe sequence: 5'[FAM]-TGCACGCGGACCAGGCATTC-[TAMRA]3', (as shown in SEQ ID NO: 4);

[0036] The reporter fl...

Embodiment 2

[0074] (3) Samples with 30

[0075] The positive plasmids in the kit were taken separately, and the concentrations were determined to be 28.2ng / μl, and the 10-fold gradient dilutions were performed with DEPC water, and a total of 5 gradients were diluted, and fluorescent RT-PCR was performed for each dilution. Obtain its slope and correlation coefficient R 2 value.

[0076] Experimental results: For the standard curve of the plasmid, see Figure 4 , whose slopes are respectively -3.357, according to the formula E (amplification efficiency) = 10 -1 / 斜率 , converting the amplification efficiency into a percentage ((E-1)×100%) is 98.6%, between 90% and 110%, indicating that the amplification efficiency of this method is good; its R 2 The values ​​are 0.996, between 0.99 and 1, indicating that the method has high reliability.

Embodiment 3

[0077] Embodiment 3 specificity test

[0078] Take 5 μL each of DNA and RNA from 5 samples of porcine blue ear virus strain, porcine pseudorabies strain, porcine parvovirus strain, BHK-21 cells, and porcine Japanese encephalitis strain as templates for fluorescent RT-PCR method For specificity detection, the plasmid plasmid was set as a positive control, the blank plasmid was used as a negative control, and DEPC water was used as a blank control group.

[0079] RT-PCR amplification conditions: 5 min at 42°C, pre-denaturation at 95°C for 10 s, cycled 40 times according to the following parameters: denaturation at 94°C for 5 sec, fluorescence collection at 60°C for 1 min. Fluorescence detection at 60°C, detection channels: FAM and TAMRA.

[0080] Fluorescent RT-PCR results show that: porcine Japanese encephalitis strain and positive control (see Figure 5 ) amplified an S-type positive curve, while the porcine blue ear virus strain, porcine pseudorabies virus strain, porcine p...

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Abstract

The invention belongs to the field of bioengineering, and provides a fluorescent RT-PCR reagent kit for detecting Japanese B encephalitis virus. The fluorescent RT-PCR reagent kit comprises a PCR reaction solution containing primers and a probe, wherein the sequence of a forward primer is as shown in SEQ ID NO:2, and the sequence of a reverse primer is as shown in SEQ ID NO:3; the sequence of the probe is as shown in SEQ ID NO:4; the 5'-end of the probe is marked with a reporting fluorophore; the 3'-end of the probe is marked with a quenching fluorophore. The invention further provides a method of adopting the reagent kit to detect the Japanese B encephalitis virus. The reagent kit disclosed by the invention can be used for quickly detecting the Japanese B encephalitis caused by the Japanese B encephalitis virus, so that the Japanese B encephalitis can be prevented and treated as soon as possible.

Description

Technical field: [0001] The invention belongs to the field of bioengineering, in particular to a detection kit, in particular to a fluorescent RT-PCR kit for detecting Japanese encephalitis virus. Background technique: [0002] Japanese encephalitis is an acute zoonotic encephalitis of central nervous system damage caused by Japanese encephalitis virus (JEV). Japanese encephalitis virus belongs to the Flavivirus genus of the Flaviviridae family, and belongs to the same genus as West Nile virus and Dengue virus. The virus can be transmitted through the bite of mosquitoes, Culex tritaeniorhynchus is the main vector. After human beings are infected with JE, the mortality rate is 20%-50%. After the disease is cured, most survivors have central nervous system sequelae. Pigs are important intermediate hosts and amplification hosts in the JEV infection process. JEV can cause reproductive disorders in sows, orchitis in boars, and cause major economic losses in the breeding industr...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6844C12Q1/70C12Q2561/101C12Q2531/113
Inventor 王建刘佩红周锦萍葛菲菲刘健鞠厚斌杨德全李鑫
Owner SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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