Development And Application Of The RT-LAMP Kit For Classical Swine Fever Virus

Classical swine fever(CSF),also known as hog cholera,is caused by the classical swine fever virus(CSFV)and is a highly fatal and contagious viral disease of worldwide importance.Indeed,it is one of the Office International des Epizobties(OIE)List A diseases.In this study,a kit for the simple and rapid detection of CSFV was established using reverse transcription loop-mediated isothermal amplification(RT-LAMP)and evaluated by the detection of clinical samples.The CSFV RT-LAMP kit was also used to determine the viral distribution and tissue tropism of the highly virulent SM strain in acutely infected pigs.To establish a specific,sensitive and rapid CSFV diagnostic technology in China and to provide a powerful method for CSF surveillance and control,a rapid assay was developed for detection of CSFV using RT-LAMP.Twelve sets of specific CSFV RT-LAMP primers were designed using 34 CSFV,18 BVDV(Bovine viral diarrhea virus)-â… and 6 BVDV-â…¡complete gene sequences downloaded from GenBank.Primers were not designed from regions showing homology with BVDV in order to guarantee CSFV-specific primers.A set of CSFV specific primers was then selected for RT-LAMP assay using RNA or DNA templates of CSFV SM,BVDV,PCV,PRRSV,PCV,PPV,PRV and FMDV. The reaction system(e.g.Betaine,Mg²⁺,dNTP,Bst DNA polymerase,AMV reverse polymerase and primers),and the reaction conditions(e.g.time and temperature)were optimized using an RNA template from CSFV SM.The CSFV RT-LAMP assay could not amplify other swine disease viruses, showing that the method has high specificity for CSFV.The CSFV RT-LAMP assay showed 10-fold higher sensitivity than the RT-nPCR assay and 10-fold lower sensitivity than the FQ-PCR assay.We evaluated the reproducibility and stability of the kit for six months using three batches of qualified RT-LAMP primers and reagents(Kit 20080829,Kit 20080916 and Kit 20081027)and finally the CSFV RT-LAMP kit is successfully built up.Twenty blind testing samples from the EU Classical swine fever reference laboratory were used to evaluated the CSFV RT-LAMP kit and the result showed that the CSFV RT-LAMP has a very high positive coincidence rate of approximately 93.33%compared with FQ-PCR and negative coincidence rate of 100%compared with FQ-PCR.Thus,due to its excellent performance,sensitivity,rapidity and low-cost,the CSFV RT-LAMP kit is more suitable for CSF surveillance and rapid detection compared to RT-PCR or FQ-PCR.To investigate the replication,dynamic distribution and tropism of the Classical swine fever virus (CSFV)in acutely infected pigs,an animal model produced using sixteen Changbai pigs that were given an intramuscular injection with 0.7ml of blood containing 4×l0^3.84TCID₅₀SM virus,and one uninfected pig as the experimental control.Clinical signs of CSF were evaluated using the scoring system suggested by Mittelholzer to follow the progression of the disease,and their body temperature were recorded daily.Two experimental pigs were killed randomly every day between day one and day eight. Eight tissues and organs,five samples for in vitro detection,blood and serum were collected from each pig and analyzed using the CSFV RT-LAMP assay.In addition,all the tissues were subjected to immunohistochemical detection.The clinical score of the infected pigs showed an increasing trend from day one to day eight post infection,which correlated well with the virus load ratio.The noneinfected control pig maintained a score of 0.Body temperature began to increase on the 2nd day after infection. The temperature remained at 41.0℃for 1.5 to two days,and then began to decline at day four to five post infection.However,there were irregular changes from day six to day eight near to death.The body temperature of the control pig remained at the normal level of 39.5℃.The result of detection by CSFV RT-LAMP was:1.Viral RNA could be detected in all the tissue samples of the infected pigs from day one post infection;2.For the in vitro samples,skin,urine and feces samples gave positive signals,all tear samples were negative,and saliva samples only gave positive signals from day four post infection; 3.All the serum samples gave positive results and one blood sample from day one post infection was negative result,while others were all positive;and 4.No viral RNA was detected using the samples from the negative control pigs.The immunohistochemical detection showed that all the tissues and organs generated positive signals,and showed an increasing trend from day one to day eight post infection.The positive signal first appeared in the capillaries and then gradually transferred into lymphocytes.No sample form the negative control pigs gave a positive signal.A total of 157 acute-phase samples with a clinical diagnosis of CSF were obtained from seven provinces of China,including blood and tonsil samples,and used to evaluate the CSFV RT-LAMP kit in comparison with RT-nPCR.All the positive products by RT-nPCR were confirmed by sequencing. CSFV RT-LAMP detected 26.11%of these samples as positive,while CSFV RT-nPCR detected 14.65% as positive.The match rate was 85.35%.This results show that the CSFV RT-LAMP kit very suitable for the detection of CSFV in clinical samples.The CSFV RT-LAMP kit is fast,easy and more sensitive than CSFV RT-nPCR.The kit will be useful for CSF surveillance and rapid detection,and has great clinical potential.