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Porcine reproductive and respiratory syndrome virus nano PCR differential diagnosis kit and detection method thereof

A respiratory syndrome and differential diagnosis technology, which is applied in the detection field of porcine reproductive and respiratory syndrome virus, can solve the problems of insufficient amplification efficiency and non-specific product amplification in complex systems, and achieve the purpose of improving specific amplification yield, The effect of reducing the residence time and shortening the time

Inactive Publication Date: 2015-12-30
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in practical applications, PCR technology has many limitations, such as non-specific product amplification and insufficient amplification efficiency in the amplification of complex systems.

Method used

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  • Porcine reproductive and respiratory syndrome virus nano PCR differential diagnosis kit and detection method thereof
  • Porcine reproductive and respiratory syndrome virus nano PCR differential diagnosis kit and detection method thereof
  • Porcine reproductive and respiratory syndrome virus nano PCR differential diagnosis kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 A kind of nano-PCR differential diagnosis kit that detects porcine reproductive and respiratory syndrome virus The best embodiment

[0033] The kit of this example includes: (1) Reagents for extracting porcine reproductive and respiratory syndrome virus RNA: TRIzolLSReagent, chloroform, isopropanol, 75% ethanol; (2) reverse transcription reagents: 5×reverse transcription buffer, concentration dNTPMixture at 2.5mM, M-MLV reverse transcriptase at a concentration of 2.5U / μL, RNase inhibitor at a concentration of 30U / μL, and reverse transcription primers; (3) Nano-PCR reaction reagents: 2×NanoPCRMix, upstream Primers, downstream primers; (4) Others: positive control and nuclease-free water. Among them, 2×NanoPCRMix consists of DNA polymerase, 2×NanoPCRbuffer and dNTPMixture, and the positive control is to inoculate Marc-145 cells with the classic strain of porcine reproductive and respiratory syndrome virus and the highly pathogenic strain respectively, and col...

Embodiment 2

[0036] Embodiment 2 Non-diagnostic purpose Distinguishing the method of porcine reproductive and respiratory syndrome virus with nano-PCR method

[0037] The detection method of this example uses the kit in Example 1. The lungs, lymph nodes, spleen, and kidneys of dead pigs were taken as samples to be tested.

[0038] The detection method of this embodiment comprises the following steps:

[0039] 1. The specific steps of virus RNA extraction are as follows: (1) After cutting the sample to be tested, add normal saline according to the mass volume ratio of 1:5 and grind evenly, centrifuge at 3000-5000rpm for 5-10 minutes, take the supernatant, and take the supernatant clear. (2) Take 250 μL of the above-mentioned treated samples and the positive control respectively, add 750 μL TRIzolLS Reagent, shake vigorously for 2 minutes, and place at room temperature for 10 minutes. Add 250 μL of chloroform, shake vigorously for 15 s, leave at room temperature for 10 min, centrifuge at ...

Embodiment 3

[0044] Example 3 Specificity Test of Porcine Reproductive and Respiratory Syndrome Virus Nano-PCR Differential Diagnosis Method

[0045] According to the method of Example 2, highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), classical strain porcine reproductive and respiratory syndrome virus (C-PRRSV), classical swine fever virus (CSFV), porcine round Cyclovirus type 2 (PCV2), porcine pseudorabies virus (PRV), and porcine boca virus (PboV) were amplified to test the specificity of the nano-PCR method. The above amplification products were analyzed by 1% agarose gel electrophoresis. The result is as figure 2 As shown, the highly pathogenic porcine reproductive and respiratory syndrome virus amplified a band of 691bp in line with the expected size, and the classical strain of porcine reproductive and respiratory syndrome virus amplified a band of 781bp in line with the expected size , while the other 4 kinds of porcine virus nucleic acid did n...

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Abstract

The invention discloses a porcine reproductive and respiratory syndrome virus nano PCR differential diagnosis kit and a detection method thereof. The kit comprises 5*reverse transcription buffering liquid, dNTP Mixture, reverse transcription inverse transcriptase, RNA enzyme inhibitor and reverse transcription primers. The method is characterized in that the kit further comprises 2*NanoPCR Mix, upstream primers and downstream primers, wherein the sequence of the upstream primers is shown as SEQ ID No.1, and the sequence of the downstream primers is shown as SEQ ID No.1. The kit can be used for detecting porcine reproductive and respiratory syndrome virus and distinguishing strain types (typical strains or highly pathogenic strains). The invention further provides a method for conducting porcine reproductive and respiratory syndrome virus detection through the kit, the detecting porcine reproductive and respiratory syndrome virus can be detected rapidly and specifically, the strain types are distinguished, the detection efficiency of the porcine reproductive and respiratory syndrome virus is greatly improved, and the specific amplification output of the porcine reproductive and respiratory syndrome virus is greatly increased.

Description

technical field [0001] The invention relates to the technical field of detection of porcine reproductive and respiratory syndrome virus. Background technique [0002] Porcine reproductive and respiratory syndrome (Porcinereproductiveandrespiratorysyndrome, PRRS), also known as porcine blue ear disease, is an infectious disease caused by porcine reproductive and respiratory syndrome virus (Porcinereproductiveandrespiratorysyndromevirus, PRRSV), with pig reproductive disorders and respiratory diseases as the main symptoms . At present, Classical porcine reproductive and respiratory syndrome virus (C-PRRSV) and Highly pathogenic porcine reproductive and respiratory syndrome virus (Highlypathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) are mainly prevalent in pig populations in my country. The main differences between the genomes of the two strains It is the deletion of 30 discontinuous amino acids in the NSP2 gene of HP-PRRSV, but there is no deletion i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/701C12Q2600/178
Inventor 袁万哲孙继国刘娜李睿文马增军王安忠左奕
Owner HEBEI AGRICULTURAL UNIV.
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