A triton X-100 assisted PMAxx-qPCR assay for rapid assessment of infectious African swine fever virus

Abstract

IntroductionAfrican Swine Fever (ASF) is a highly infectious disease of pigs, caused by African swine fever virus (ASFV). The lack of vaccines and drugs makes strict disinfection practices to be one of the main measurements to curb the transmission of ASF. Therefore, it is important to assess if all viruses are inactivated after disinfection or after long time exposure in their natural conditions. Currently, the infectivity of ASFV is determined by virus isolation and culture in a biosafety level 3 (BSL-3) laboratory. However, BSL-3 laboratories are not readily available, need skilled expertise and may be time consuming.MethodsIn this study, a Triton X-100 assisted PMAxx-qPCR method was developed for rapid assessment of infectious ASFV in samples. PMAxx, an improved version of propidium monoazide (PMA), can covalently cross-link with naked ASFV-DNA or DNA inside inactivated ASFV virions under assistance of 0.1% (v/v) TritonX-100, but not with ASFV-DNA inside live virions. Formation of PMAxx-DNA conjugates prevents PCR amplification, leaving only infectious virions to be detected. Under optimum conditions, the limit of detection of the PMAxx-qPCR assay was 2.32log10HAD50/mL of infectious ASFV. Testing different samples showed that the PMAxx-qPCR assay was effective to evaluate intact ASFV virions after treatment by heat or chemical disinfectants and in simulated samples such as swine tissue homogenate, swine saliva swabs, and environmental swabs. However, whole-blood and saliva need to be diluted before testing because they may inhibit the PCR reaction or the cross-linking of PMAxx with DNA.ConclusionThe Triton X-100 assisted PMAxx-qPCR assay took less than 3 h from sample to result, offering an easier and faster way for assessing infectious ASFV in samples from places like pig farms and pork markets.

Current detection methods of African swine fever virus

“…With heat evaluation treatments, the detection limit for ASFV is 10 2.28 HAD50/mL, while with chlorine disinfectants, the detection limit is 10 5.28 HAD50/mL. Furthermore, Liu et al (46) has developed a Triton X-100-assisted PMA-qPCR method that allows for the assessment of ASFV infectivity in samples, with a LOD of 10 2.32 HAD50/mL.…”

Application of propidium monoazide quantitative PCR to discriminate of infectious African swine fever viruses

“…In experimental bacterial strains, PMAxx increased the difference between live and dead bacteria by an additional 3 to 7 CT values compared to PMA ( Nocker et al, 2006 ). Recently, Liu et al conducted a study to investigate the addition of Triton X-100 for enhancing the penetration of PMAxx into inactivated ASFV virions, which may be helpful to interpret the results, though the infectivity of samples was still distinguishable by PMAxx-qPCR without the assistance of Triton X-100 ( Liu et al, 2022 ). Moreover, these experiments were carried out solely under laboratory conditions and the validity of the results was not confirmed through animal inoculation.…”