Establishment And Evaluation Of A Cross-priming Amplification Assay For Detecting Pseudorabies Virus

Pseudorabies?PR?,also called as Aujeszky's disease,is an acute infectious disease of pigs and some mammals caused by pseudorabies virus?PRV?.The disease can cause pregnant sows reproductive disorders,newborn piglets death and adult pigs latent infection.Latently infected pigs do not show typical clinical symptoms,but life-long poisoning and cyclical detoxification increase the difficulty of eradication.Since late 2011,national-wide PR outbreaks are increased in many Bartha-K61-vaccinated swine herds in China,and the epidemic has been confirmed by the PRV mutation.Thus,a fast,convenient and on-site diagnosis is essential for surveillance to reduce the spread of the disease.Nucleic acid isothermal amplification?NAIA?is a new type of molecular diagnostic techniques that carried out at one constant temperature.This feature simplifies the requirements of amplification equipment and shortens the reaction time,making it ideal technology for on-site detection.Cross-priming amplification?CPA?is China's first isothermal expansion technology with independent intellectual property rights.CPA combined with immunochromatographic strip can be widely used in molecular diagnostics,epidemic prevention and quarantine,biomedical research and other fields.In addition to Bst DNA polymerase,there are amplification primers and cross primers in the amplification system.The target fragment relies on the high-activity strand displacement of Bst DNA polymerase,which allows the DNA cycle to be continuously realized.This study developed and evaluated a cross-priming amplification in combination with immunochromatographic strip assay?CPA-strip?for specific detection of wild-type PRV?variant and classical strains?,which can be used for early diagnosis.Objective:This study aims to establish a CPA method for the detection of PRV,which rapid,sensitive and easy-to-judge.Methods:The missing gI/gE gene region of the Bartha-K61 vaccine strain was used for primers and probes design to detect classical or variant PRV strains based on a DNA sequence alignment of BLAST analysis on the NCBI website.The initial amplification system was established,and the parameters in the reaction system were optimized to select the optimal primers from many pairs of primers.The established method,which was determined as the optimum reaction system,was then combined with immunochromatographic strip for sensitivity,specificity and reproducibility tests.The actual samples were verified by triplex real-time PCR and virus isolation test for compliance analysis.Results:The final reaction system was as follows:the 25-?L reaction mixture contained 2.0?L of PRV-1s-2a?10?M?,0.5?L of PRV-4a and PRV-5s?10?M?,1.5?L of PRV-3s and PRV-1s?10?M?,2.5?L of dNTPs?10 mM?,1.0?L of Mg²⁺?100mM?,2.5?L of ThermoPol buffer?10×?,1.0?L of Bst DNA polymerase?8 U/?L?,2.5?L of betaine,2.0?L of genomic DNA and nuclease-free water to 25.0?L.The mixture was incubated at 61°C for 60 min.The reaction products were analyzed by1.5%agarose gel electrophoresis.The sensitivity of the assay was determined to be2.0×10² copies for the recombinant plasmid of PRV variant strain.Specificity test result showed that the CPA-strip assay had no cross-reactivity with several non-PRV porcine viruses.Repetitive test result showed that the color intensity among the test lines was the same in inter-and intra-assay among the test lines.When testing a total of 293 clinical field swine samples,the agreement among CPA-strip,triplex real-time PCR and virus isolation were 100%?293/293?and 97.3%?285/293?for wild-type PRV strains,and six samples positive for the Bartha-K61 vaccine strains showed negative results in the CPA-strip assay.In summary,this method,which combines the efficient DNA amplification ability of CPA with the visualization characteristic of test strips,is simple and reliable,no special equipment,and has good sensitivity and specificity.It can be used for PRV detection and applies to the current increasingly serious PR epidemic.