A Taqman real-time fluorescent PCR kit for detecting pig umbilical cord blood porcine transmissible gastroenteritis virus and its application

Abstract

The invention discloses a Taqman real-time fluorescent PCR kit for detecting pig umbilical cord blood transmissible gastroenteritis virus and application thereof. The kit comprises a pair of primers and a specific fluorescent probe, the sequences of the primers are shown as SEQ ID NO:1 and SEQ ID NO:2, and the sequence of the specific fluorescent probe are shown as SEQ ID NO:3. The Taqman real-time fluorescent PCR kit can specifically detect the pig umbilical cord blood transmissible gastroenteritis virus, and has high sensitivity, strong capacity of resisting disturbance, strong universality and good repeatability. The kit can precisely evaluate and diagnose conditions of carrying and expelling of porcine transmissible gastroenteritis virus of sows as well as infection of piglets, can further implement purification and effect evaluation of pig transmissible gastroenteritis, and has a very high application value. Meanwhile, the kit has a simple and convenient detecting process, needed instruments and equipment are simple, and detecting time is short; the result is relatively reliable, and the kit is not prone to contamination; the kit has a low technological operation demand, and can be generally popularized in the grass root; quality control is relatively easy, and the kit is easy to standardize.

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 pig umbilical cord blood porcine transmissible gastroenteritis virus and its application. Background technique [0002] Transmissible gastroenteritis of swine (TGE) is a porcine diarrheal disease caused by porcine transmissible gastroenteritis virus (TGEV). Infectious disease, which can cause severe diarrhea and vomiting in piglets under 2-3 weeks of age, and lower morbidity and mortality in pigs over 5 weeks of age. The disease has widely existed in Europe, America, and Asian countries, and was first discovered in pigs in my country in the 1960s. In my country, the disease mainly occurs in two forms of outbreak and endemicity. Since 2010, the prevalence and degree of harm have deepened day by day. TGEV often co-infects with porcine epidemic diarrhea virus (PEDV) and porcine rotavirus (PoRV), which brings difficul...

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Problems solved by technology

[0005] However, the above serological diagnostic methods have the following disadvantages: (1) It is difficult to evaluate the immune tolerance phenomenon in pig herds, and they cannot reflect the antibody level of pig herds more effectively, intuitively and accurately

(2) It cannot be quantified, and false positives are prone to occur, and blood sample collection requires the assistance of multiple people and special equipment, and the collection process is relatively time-consuming

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

[0006] However, molecular diagnostic methods, regardless of the common PCR and fluorescent PCR methods, have the following defects and deficiencies: (1) the detection process is complicated, the technical requirements are high, false positives are prone to occur, and the environment is seriously polluted; (2) molecular diagnostics have strict requirements on sample tissue tropism , such as: feces or intestines need to be collected for infectious gastroenteritis disease materials, and the requirements for collecting samples are strict; (3) This diagnostic method can only evaluate the situation of this sample, but cannot simultaneously and accurately diagnose the situation of two pig herds, etc.; ( 4) In addition to the above shortcomings, ordinary PCR cannot be quantified, and the operation is cumbersome, etc.

However, due to the above defects in the common PCR or fluorescent PCR methods currently used, the diagnostic results have certain limitations in the guiding significance of disease prevention and control in pig farms.

[0008] In actual production, because the TGEV virus cannot adapt to the cells well, and the cytopathic changes are not obvious, it is difficult to isolate the virus; the clinical symptoms of TGEV infection and PEDV infection are almost the same, and it is difficult to make a clinical differential diagnosis; but the use of fluorescence in umbilical cord blood Detection of TGEV by PCR detection technology is the most effective diagnostic method for early warning, diagnosis and evaluation of diseases, but it is relatively difficult to quickly and accurately detect porcine transmissible pneumoenteritis virus in umbilical cord blood, because 1) hemolytic factors have a great impact on the detection effect , 2) TGEV virus belongs to single-stranded positive-strand RNA virus, easy to mutate, 3) regardless of PCR and fluorescent PCR technology, there are defects in the above methods

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