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Kit for simultaneously detecting classical strains and variant strains of porcine reproductive and respiratory syndrome virus and detection method thereof

A technology for respiratory syndrome and virus mutation, applied in the direction of microorganism-based methods, biochemical equipment and methods, measuring devices, etc., can solve the problems of high experimental cost, high variability, and long time consumption, and achieve high sensitivity and rapid Accurate detection and good specificity

Active Publication Date: 2010-12-29
山东兴安智慧科技有限公司
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

One type is step-by-step detection, and only one of them can be detected at a time. For example, the patent "RT-PCR Kit for Porcine Reproductive and Respiratory Syndrome Virus Super Mutant Strain" [200710086548] of the China Animal Disease Prevention and Control Center, this type of The method has the characteristics of repetitive operation, long time-consuming, high experimental cost, easy to introduce human error, and not suitable for grass-roots applications.
The other is the method of identifying RT-PCR. For example, the method established by Hao Xiaofang et al. uses NSP2 as the only detection target gene, and uses the amplification size of the target band as a standard to identify PRRSV mutant strains and classic strains [highly pathogenic pig reproduction and The establishment of RT-PCR differential diagnosis method for respiratory syndrome virus, Chinese Journal of Preventive Veterinary Medicine; 2007, 29 (5): 704-709.], the double PCR established by He Dan et al. NSP2 mutant strain [Establishment and application of double PCR diagnostic method for porcine reproductive and respiratory syndrome virus American strain and NSP2 mutant strain, Guangxi Agricultural Sciences 2009, 40(1): 84-87.], this kind of method uses NSP2 as the only Detect the target gene, but NSP2 is a hypervariable region in the PRRSV genome, which has high variability and brings potential instability to the experimental results

Method used

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  • Kit for simultaneously detecting classical strains and variant strains of porcine reproductive and respiratory syndrome virus and detection method thereof
  • Kit for simultaneously detecting classical strains and variant strains of porcine reproductive and respiratory syndrome virus and detection method thereof
  • Kit for simultaneously detecting classical strains and variant strains of porcine reproductive and respiratory syndrome virus and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Preparation of the kit.

[0048] 1 virus strain:

[0049] 1.1 Preparation of negative control samples

[0050] Take sterilized DEPC water and store it in a -20°C refrigerator.

[0051] 1.2 Preparation of Positive Control Samples of Porcine Reproductive and Respiratory Syndrome Virus Mutants:

[0052] 1.2.1 Preparation of RPMI-1640 medium:

[0053] One bag of RPMI-1640 powder (10.4g, purchased from Invitrogen Company), dissolved in 1000mL three-distilled water, added Hepes 5.96g, sodium pyruvate 0.11g, NaHCO 3 2.2g, after fully dissolved by stirring, N 2 Sterilize by positive pressure filtration and store at 4°C for later use.

[0054] 1.2.2 Preparation of reagents for cell culture:

[0055] (1) Double Antibody: Weigh 1 g of penicillin and 6.42 g of streptomycin, dissolve in 160 mL of double distilled water, filter and sterilize with a 0.22 μm filter, and store in -20°C after aliquoting.

[0056] (2) 0.2% trypsin solution: Weigh 0.2 g of trypsin, disso...

Embodiment 2

[0107] Embodiment 2: detection method.

[0108] Utilize the kit of embodiment 2 to detect the method for porcine reproductive and respiratory syndrome virus (PRRSV) classic strain and mutant strain simultaneously, comprise the steps:

[0109] (1) Extraction of RNA:

[0110] (1a) Take 0.5g of the tissue to be tested, place it in a homogenizer or a mortar and grind it, and dilute it with 1.5mL D-Hanks solution; take the ground tissue fluid to be tested, put it in a 1.5mL sterilized centrifuge tube, and centrifuge at 6000rpm 5min;

[0111] (1b) Take 300 μL of the supernatant obtained by centrifugation in step (1a) into a 1.5 mL sterilized centrifuge tube, add 1 mL of Trizol to mix, and place at room temperature for 10 minutes; then add 200 μL of chloroform to mix, and place at room temperature for 2 minutes;

[0112] (1c) Centrifuge the sterilized centrifuge tube in step (1b) at 12,000 rpm for 10 min, take 600 μL of the upper aqueous phase into another 1.5 mL sterilized centrif...

Embodiment 3

[0126] Embodiment 3: specificity test.

[0127] 1 material

[0128] 1.1 Sample

[0129] 1.1.1 Other porcine susceptible virus strains: Japanese encephalitis virus (JEV), swine fever virus (CSFV), porcine parvovirus (PPV), porcine pseudorabies virus (PRV), porcine circovirus (PCV) , Porcine transmissible gastroenteritis virus (TGEV), kept by the unit.

[0130] 1.1.22 SPF porcine lungs and serum.

[0131] 1.1.3 North American reference strain of PRRSV (VR2332 strain).

[0132] 2 methods

[0133] 2.1 Specificity test method

[0134] 6 other porcine susceptible virus strains, 2 SPF pig lungs and serum were detected with the novel porcine reproductive and respiratory syndrome virus (PRRSV) RT-PCR detection kit. Check the specificity of the kit for detecting different samples.

[0135] 3 results

[0136] 3.1 Detection of other porcine susceptible virus strains, SPF pig samples and other porcine reproductive and respiratory syndrome virus results

[0137] The new porcine rep...

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Abstract

The invention discloses primer pairs for simultaneously detecting classical strains and variant strains of porcine reproductive and respiratory syndrome virus, which have nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. The invention also discloses a kit which comprises the primer pairs for simultaneously detecting the classical strains and the variant strains of the porcine reproductive and respiratory syndrome virus, and a method for simultaneously detecting the classical strains and the variant strains of the porcine reproductive and respiratory syndrome virus by adopting the kit. The primers in the kit for detecting the porcine reproductive and respiratory syndrome virus have the advantages of higher specificity, sensitivity and stability, and capacity of providing a rapid, sensitive and specific method for detecting the porcine reproductive and respiratory syndrome virus.

Description

technical field [0001] The invention belongs to the field of animal protection and quality inspection, and relates to a porcine reproductive and respiratory syndrome virus detection kit and a detection method thereof, in particular to a porcine reproductive and respiratory syndrome virus RT-PCR detection kit and a detection method thereof. Background technique [0002] In the summer and autumn of 2006, Jiangxi, Hunan, Hubei, Anhui and Jiangsu Nanjing, Wuxi, Yangzhou, Suzhou and other major domestic pig-raising areas will have an infectious disease characterized by high fever, loss of appetite, dyspnea and reproductive disorders. The cause of the disease has not yet been clarified. According to the clinical characteristics of the elevated body temperature of sick pigs, the disease is called "unknown high fever in pigs" or "high fever disease" in pigs. High fever spread widely and lasted for several months. 13 to 25 week-old growing and finishing pigs are more frequent. The ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11G01N27/447C12R1/93
Inventor 张志成范红结陈钟鸣戴鼎震方光远何小明
Owner 山东兴安智慧科技有限公司
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