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Mutiplex PCR detection kit for detecting PRRSV, PCV2, PRV and CSFV

A kit and multiple technology, applied in the field of in vitro molecular diagnosis of biomedicine, can solve the problems of low sensitivity and specificity, time-consuming, labor-intensive and material-consuming, complicated operation, etc., and achieve the effect of specific detection

Inactive Publication Date: 2016-04-20
邓小红
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Because these diseases often present similar symptoms clinically, and mixed infections often occur, and it is difficult to make a differential diagnosis based on clinical symptoms, conventional serological diagnosis methods are complicated to operate, time-consuming, labor-intensive, material-intensive, sensitive and specific. Compared with the traditional method, the PCR method is fast, sensitive and easy to operate, and is widely used in the detection and differential diagnosis of infectious diseases in livestock and poultry. Many scholars have established the pathogenic method for these four pathogens PCR diagnostic method, but when infected with multiple pathogens, different systems and procedures make the single-plex PCR method have many steps and a large amount of actual use

Method used

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  • Mutiplex PCR detection kit for detecting PRRSV, PCV2, PRV and CSFV
  • Mutiplex PCR detection kit for detecting PRRSV, PCV2, PRV and CSFV
  • Mutiplex PCR detection kit for detecting PRRSV, PCV2, PRV and CSFV

Examples

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Effect test

Embodiment 1

[0034]Example 1 Detects the construction of multiple PCR kits for porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, porcine pseudorabies virus and classical swine fever virus

[0035] The kit of this embodiment includes: (1) RNA extraction reagents: TRIzolLSReagent, chloroform, isopropanol, 75% ethanol; (2) DNA extraction reagents: proteinase K, lotion, absolute ethanol; (3) reverse transcription reagents : 5× reverse transcription buffer, dNTPMixture with a concentration of 2.5mM, M-MLV reverse transcriptase with a concentration of 2.5U / μL, RNase inhibitor and reverse transcription primer with a concentration of 30U / μL; (4) PCR reaction reagents: 2×PCRMix, upstream primers, downstream primers; (5) Others: positive control and nuclease-free water. Among them, 2×PCRMix is ​​composed of DNA polymerase, 2×PCRbuffer and dNTPMixture. The positive control is to inoculate the cells with virus and harvest the cell culture. The primers are lyophilized powd...

Embodiment 2

[0038] Embodiment 2 The construction of the method for detecting porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, porcine pseudorabies virus and classical swine fever virus by multiplex PCR

[0039] The detection method of this example uses the kit in Example 1. Take the lungs, lymph nodes, and spleen. Kidney is the sample to be tested.

[0040] The detection method of this embodiment comprises the following steps:

[0041] 1. The specific steps of viral RNA extraction are as follows: (1) After cutting the sample to be tested, add physiological saline according to the mass volume ratio of 1:5 and grind evenly, centrifuge at 3000~5000rpm for 5~10 minutes, and take the supernatant. (2) Take 250 μL of the above-mentioned treated sample and 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 tempe...

Embodiment 3

[0048] Embodiment 3 sensitivity test

[0049] The nucleic acid templates with total nucleic acid concentrations of 48.1ug, 29.6ug, 54.2ug and 62.8ug of PCV2, PRV, PRRSV and CSFV were measured by ultra-micro spectrophotometer for sensitivity test.

[0050] The PCR amplification product was detected by 1% agarose gel electrophoresis, and the results were as follows: figure 2 shown. It can be seen that the minimum detection amounts of PCV2, PRV, PRRSV, and CSFV by multiplex PCR were 48.1pg, 29.6pg, 54.2pg, and 62.8pg, respectively.

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Abstract

The invention provides a mutiplex PCR detection kit for detecting PRRSV, PCV2, PRV and CSFV. The mutiplex PCR detection kit comprises an inverse transcription system, 2XNanoPCR Mix and a primer combination, wherein the inverse transcription system is 5X inverse transcription buffer solution, dNTP Mixture, reverse transcriptase, an rnaase inhibitor and an inverse transcription primer, and the primer combination is shown as SEQ ID NO.1-8. The mutiplex PCR detection kit can be used for detecting the porcine reproductive and respiratory syndrome virus, the porcine circovirus type 2, the porcine pseudorabies virus and the classical swine fever virus. The invention further provides a method for detecting the four viruses by adopting the kit, the method can be used for rapidly and specifically detecting the porcine reproductive and respiratory syndrome virus, the porcine circovirus type 2, the porcine pseudorabies virus and the classical swine fever virus, and the detection efficacy of the four viruses is greatly improved.

Description

technical field [0001] The invention belongs to the technical field of in vitro molecular diagnosis of biomedicine, and in particular relates to a multiplex PCR detection kit for PRRSV, PCV2, PRV and CSFV. Background technique [0002] Porcine reproductive and respiratory syndrome (Porcinereproductiveandrespiratorysyndrome, PRRS) is caused by porcinereproductiveandrespiratorysyndromevirus (Porcinereproductiveandrespiratorysyndromevirus, PRRSV), an infectious disease with pig reproductive disorders and respiratory system diseases as the main symptoms. Circovirus-associated diseases (PCVAD) are a series of related clinical diseases caused by circovirus type 2 (PCV2), including postweaning multisystemic wasting syndrome (Postweaning multisystemic wasting syndrome, PMWS), porcine dermatitis and Nephrotic syndrome (Porcinedermatitisandnephropathysyndrome, PDNS) and porcine respiratory disease syndrome (Porcinerespiratorydiseasesyndrome, PRDS) and so on. Among them, PMWS is the m...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/70C12Q1/686C12Q2537/143C12Q2521/107
Inventor 邓小红袁万哲张军余伟权李桃梅李彬
Owner 邓小红
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