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Porcine fulminating infectious disease pathogen multiplex detection kit and application thereof

A detection kit and infectious disease technology, applied in the field of molecular biology, can solve the problems of low detection throughput, low sensitivity, and poor sensitivity, and achieve the effects of simplifying experimental operation steps, preventing environmental pollution, and simple application methods

Pending Publication Date: 2022-04-15
无锡科智达科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Porcine severe infectious diseases pose a serious threat to the pig breeding industry, such as African swine fever, porcine reproductive and respiratory syndrome, classic swine fever and porcine pseudorabies and other severe infectious diseases pose a great threat to the pig industry, and also give pigs caused serious economic losses
Porcine severe infectious diseases are highly contagious, morbidity and lethal, similar to other non-severe infectious diseases in clinical symptoms, clinical symptoms are not easy to distinguish, and often mixed with other pathogens, which brings great trouble to clinical diagnosis
At present, laboratory diagnostic methods mainly include pathogen direct smear microscopic observation, cell culture examination, serum enzyme-linked immunological detection and nucleic acid detection technology, among which the specificity of microscopic examination and culture method is the best, but the sensitivity is poor. Collection, storage, transportation, and experimental conditions are demanding, and the detection cycle is long. Some pathogens are extremely difficult to cultivate and quality control is impossible, and the detection rate of mixed infections is low; while the sensitivity of immunological detection is low, and pathogen infection has a window period. The diagnostic value of severe infectious diseases of swine with high mortality rate is not great
Nucleic acid detection technology has been more and more widely used in the detection of swine disease-related pathogens due to its high sensitivity and specificity, but the low detection throughput and the need for cold chain transportation and low temperature storage of detection reagents limit its application in pathogenic microorganism Aspects of application
Therefore, the above methods have certain limitations in practical application.

Method used

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  • Porcine fulminating infectious disease pathogen multiplex detection kit and application thereof
  • Porcine fulminating infectious disease pathogen multiplex detection kit and application thereof
  • Porcine fulminating infectious disease pathogen multiplex detection kit and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Porcine severe infectious disease pathogen multiplex detection kit: the kit includes specific primers for amplifying 4 gene loci of African swine fever virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus and porcine pseudorabies virus and fluorescent probes; among them, 4 gene loci are: VP72 gene of African swine fever virus, N gene of porcine reproductive and respiratory syndrome virus, 5'UTR gene of classical swine fever virus, gD gene of porcine pseudorabies virus ; and specific primers and fluorescent probes for pig internal reference gene ACTB.

[0021] The sequences of the specific primers and fluorescent probes are as follows: ASFV, SEQ ID NO.1-3; PRRSV, SEQ ID NO.4-6; CSFV, SEQ ID NO.7-9; PRV, SEQ ID NO.10-12 ; Sus, SEQ ID NO. 13-15.

[0022] Table 1 Specific primers and fluorescent probe sequence list

[0023]

[0024] The kit includes PCR reaction solution I and PCR reaction solution II; wherein, PCR reaction solution I...

Embodiment 2

[0028] Embodiment 2 specificity test

[0029] (1) The detection device consists of two parts, one part is the extraction reagent, which can be filled with the corresponding volume of extraction reagent according to the requirements, and sealed in plastic; the other part is the detection reagent, which is in the form of freeze-dried balls, mainly composed of primers, Mg 2+, dNTPs, polymerase, reverse transcriptase, etc., the components are prepared in a certain proportion and then freeze-dried. The freeze-drying procedure includes pre-freezing at -45-50°C, primary drying, and secondary drying. After leaving the compartment, it is combined with the extraction reagent part and stored in vacuum.

[0030] (2) Take out the detection device, put the plasmid containing the African swine fever virus VP72 gene sequence, Keqian pig blue ear live vaccine (JXA1-R strain), Qilu swine fever live vaccine, Keqian pseudorabies live vaccine (HB-98 strain) ), Harvey porcine transmissible gastro...

Embodiment 3

[0036] Embodiment 3 Sensitivity detection test

[0037] Dilute the plasmid DNA containing the respective targets of African swine fever virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus and porcine pseudorabies virus with sterile purified water, and then add them to the detection device respectively. detection on the detection hole.

[0038] It can be shown from the test results that the detection sensitivity of the detection method to African swine fever virus, porcine reproductive and respiratory syndrome virus, classical swine fever virus and porcine pseudorabies virus is 500 copies / reaction.

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Abstract

The invention discloses a porcine fulminating infectious disease pathogen multiplex detection kit and application thereof. The kit comprises specific primers and fluorescent probes for amplifying four gene loci of an African swine fever virus, a porcine reproductive and respiratory syndrome virus, a classical swine fever virus and a porcine pseudorabies virus. Compared with the prior art, the invention has the following advantages: (1) a multiplex fluorescent quantitative PCR amplification technology and a freeze-drying technology are combined, simultaneous detection of multiple indexes of a single sample is realized, the system simplifies experimental operation steps, improves detection flux, provides a new method for accurate detection of multiple indexes of pathogens of porcine fulminating infectious diseases, and has good application prospects in the field of detection of multiple indexes of pathogens of porcine fulminating infectious diseases. The urgent requirements of multi-index rapid screening and accurate detection are met; (2) in the detection process, the detection areas are completely isolated from each other and sealed, and after detection is completed, the sealing reagent is cured, so that outward diffusion of amplification products in the whole detection process and after detection is completed is effectively prevented, and environmental pollution is prevented.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to the detection of four kinds of pig infectious disease pathogens, in particular to a multiple detection kit for severe infectious disease pathogens of pigs and an application thereof. Background technique [0002] Acute and severe infectious diseases of pigs are extremely contagious and pathogenic, and the mortality rate is extremely high. Such as African swine fever, swine fever, foot-and-mouth disease, erysipelas, pneumonia and other major diseases. [0003] Porcine severe infectious diseases pose a serious threat to the pig breeding industry, such as African swine fever, porcine reproductive and respiratory syndrome, classic swine fever and porcine pseudorabies and other severe infectious diseases pose a great threat to the pig industry, and also give pigs caused serious economic losses. Severe porcine infectious diseases are highly contagious, morbidity, and lethal. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
Inventor 唐大运陈昌海陈俊飞邓永杰刘梅
Owner 无锡科智达科技有限公司