A Taqman real-time fluorescent PCR kit for diagnosing pig umbilical cord blood pseudorabies virus field strain and its application

Abstract

The invention discloses a Taqman real-time fluorescence PCR kit for diagnosis of wild strain of pseudorabies virus in pig umbilical cord blood and application thereof. The kit comprises a pair of primers and a specific fluorescence probe. Sequence of the primers is as shown in SEQ ID NO:1 and SEQ ID NO:2. Sequence of the fluorescence probe is as shown in the SEQ ID NO:3. The kit provided by the invention can specifically diagnose wild strain of pseudorabies virus in pig umbilical cord blood, has high sensitivity and excellent repeatability, can accurately evaluate and diagnose sows situations of carrying and eliminating pseudorabies virus and piglet infection situations, can be used for carrying out purification and effectiveness evaluation of pseudorabies disease, and has very wide application value. Meanwhile, the kit requires relatively simple instruments and equipment during the detection process, the detection process is simple, and detection time is short. Results are relatively reliable, and the kit is not easy to pollute. As technical operation requirement is low, the kit can be promoted in quantity at the grassroots. The kit is relatively easy for quality control and easy 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 diagnosing wild strains of pig umbilical cord blood pseudorabies virus and its application. Background technique [0002] Porcine pseudorabies virus (PRV) can cause pseudorabies (Pesudorabies, PR) in a variety of domestic and wild animals. The disease is extremely harmful to pigs, and the main clinical symptoms are abortion, mummification, stillbirth and weak offspring in pregnant sows. For suckling piglets and weaning piglets, there will be high fever, dyspnea and obvious symptoms of central nervous system disorders, and the mortality rate after infection can reach 100%. [0003] Currently commonly used diagnostic methods for swine pseudorabies: (1) serum neutralization test (serum neutralization test, SN), which is one of the main methods for detecting pseudorabies in most countries. SN has strong specificity and low sens...

AI Technical Summary

Problems solved by technology

Among the above four methods, the specificity and sensitivity of serum neutralization test are higher than other detection methods, but due to its high requirements and long cycle, it is not as simple as ELISA test and latex agglutination test in the detection of large quantities of samples fast

At the same time, serological detection techniques (SN, LAT and ELISA) have the following disadvantages: (1) It is difficult to evaluate the immune tolerance phenomenon in pigs, and it is relatively impossible to reflect the antibody level of pigs more effectively, intuitively and accurately

(2) Blood sample collection requires the assistance of multiple people and special equipment, and the collection process is relatively time-consuming

PCR technology is fast and sensitive, but due to the high GC content of pseudorabies gE gene, it is relatively difficult to amplify, and the gene cannot be detected in general clinical test samples (in umbilical cord blood), so it is impossible to use ordinary PCR technology for disease diagnosis of wild strains

In addition, PCR detection of pseudorabies samples 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: it is very easy to contaminate, and even domestic authoritative laboratory test results have false positive results

(4) High environmental 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) Each laboratory independently designs primers, the detection sensitivity is inconsistent, the quality control is not in place, the standards are not uniform, and it is difficult to standardize and analyze data

(6) The tissue tropism of the virus is different, and sampling is difficult

[0004] At present, serological (ELISA) method is commonly used to diagnose wild strains of pseudorabies virus, but the serological detection method is antigen and antibody reaction, and only one detection cannot accurately determine the infection status and detoxification status of pigs; serological detection of antibodies, the detection of Antibody levels cannot be quantitatively detected and analyzed, and serology can only evaluate the level of humoral immunity; there are technical bottlenecks in serological evaluation of pig herd health, and evaluation of asymptomatic virus-carrying and immune-tolerant pig herds; serological needs Collecting blood from the anterior vena cava, blood sample collection is relatively laborious, time-consuming, and requires special equipment assistance, multi-person assistance

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