A Taqman real-time fluorescent PCR kit for detecting wild strains of porcine epidemic diarrhea virus in pig umbilical cord blood and its application

Abstract

The invention discloses a Taqman real-time fluorescent PCR (Polymerase Chain Reaction) kit for detecting porcine epidemic diarrhea viruswild strains in porcine umbilical cord blood and use of the PCR kit. The kit comprises a pair of primers and a specific fluorescent probe, wherein sequences of the primers are as illustrated by SEQ ID NO: 1 and SEQ ID NO: 2, and a sequence of the fluorescent probe is as illustrated by SEQ ID NO: 3. The kit disclosed by the invention can be used to perform specific qualitative and quantitative detection on the porcine epidemic diarrhea viruswild strains in the porcine umbilical cord blood, has the advantages of higher hemolysis resistance, pollution prevention, sensitivity, good universality and excellent repeatability, can be used to accurately evaluate and diagnose existence, elimination and piglet infection conditions of the porcine epidemic diarrhea viruswild strains of a sow, also can be used to carry out evaluation on effect of a porcine epidemic diarrhea vaccine, and therefore has a very wide range of application value. Meanwhile, the kit has the advantages of a simple and convenient detection process, simplicity in required instruments and equipment, short detection time, relatively reliable result, no pollution, low requirement on technical operations, capability of being widely popularized in the basic layer, relative easiness in quality control and easiness for standardization.

Description

technical field [0001] The invention relates to the technical field of molecular diagnosis, in particular to a Taqman real-time fluorescent PCR kit for detecting wild strains of porcine epidemic diarrhea virus in pig umbilical cord blood and its application. Background technique [0002] Porcine epidemic diarrhea virus (PEDV) can cause porcine epidemic diarrhea (porcine epidemic diarrhea, PED), a highly contagious intestinal infectious disease, the main clinical symptoms are diarrhea, vomiting, dehydration, High morbidity and high mortality, other age pigs mainly showed transient diarrhea, low mortality. PEDV belongs to the αcoronavirus genus of the Coronaviridae subfamily of the Coronaviridae family. The virus is slightly spherical in shape and pleomorphic in feces. PEDV particles are very similar to virions of other coronaviruses. They have an envelope with petal-shaped fibrils that radiate from the core image. The distance between them is large and the arrangement is reg...

AI Technical Summary

Problems solved by technology

[0006] However, the above-mentioned serological detection technology has the following disadvantages: (1) It is difficult to evaluate the phenomenon of immune tolerance in pig herds, wild virus and immune distinction, and it is relatively unable to reflect the antibody level of pig herds more effectively, intuitively and accurately

(2) The blood sample collection process is relatively time-consuming and complicated

The reasons are as follows: ①The serological detection method is antigen and antibody reaction, and the infection status and detoxification status of pigs cannot be accurately judged only by one detection; Evaluate the level of humoral immunity in the body; ③ There are technical bottlenecks in the serological assessment of the health of pigs and the assessment of asymptomatic virus-carrying and immune-tolerant pigs; ④ Serology requires the collection of anterior vena cava blood, and blood sample collection is relatively laborious , time-consuming, and requires special equipment assistance, multi-person assistance

[0007] The common PCR method for molecular diagnosis also has the following disadvantages: (1) complex instruments and equipment are required: nucleic acid extraction instrument, PCR instrument, electrophoresis instrument, gel imaging system and analysis software

(2) The operation is complicated and the operation takes a long time: nucleic acid extraction, PCR reagent configuration, PCR reaction, electrophoresis, agglutination imaging, analysis results, etc. are required, and it takes nearly a day to do a pathogen detection

(3) The results are relatively unreliable: aerosols are easily formed during the operation process, leading to environmental pollution. Even domestic authoritative laboratory test results have false positive results

(4) High technical operation requirements: it is difficult to promote at the grassroots level, pig farms are equipped with equipment but no one and time to use it

(5) Poor anti-interference ability to hemolyzed samples, affecting the amplification effect, (6) Relatively low sensitivity compared with fluorescent PCR, and cannot be quantified

However, because cord blood samples are easily hemolyzed during collection and transportation, and hemolysis has a great impact on PCR amplification, and the sensitivity and specificity of PCR primers and probes currently used in clinical practice are inconsistent, the environmental pollution of routine laboratories is serious, and direct Affect the detection results, and then affect the prevention and control of PEDV diseases

[0009] Although qPCR can overcome the above problems, due to the high sensitivity of qPCR, aerosols are easily formed during the nucleic acid extraction process, and false positives are prone to occur. In addition, because PEDV is an RNA virus, it is easy to mutate, and there is hemolysis in blood samples, which seriously affects qPCR. How to screen more sensitive and specific primers and probes and amplification techniques, so as to solve the above problems, is the focus of the present invention's research

[0010] In addition, in actual production, it is relatively difficult to quickly and accurately detect wild strains of porcine epidemic diarrhea virus in umbilical cord blood. Wild strains of PEDV have low adaptability to cells and do not produce obvious cell lesions. It is difficult to isolate the virus. Moreover, the clinical symptoms of porcine epidemic diarrhea virus are the same as those of porcine transmissible gastroenteritis (TGEV) virus, and it is difficult to distinguish them. For the diagnosis and control of the disease, it is necessary to establish a rapid differential diagnosis of porcine epidemic diarrhea virus field strains in umbilical cord blood. The method can not only overcome the interference of umbilical cord blood hemolysis, but also can evaluate the safety of PEDV vaccine strains in sows, quickly and accurately diagnose PEDV virus detoxification in sows and embryo infection in piglets, and formulate timely prevention and control measures for PEDV disease early warning and prevention. control plan, which has very important clinical significance

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