Polymerase cross-linking spiral reaction (PCLSR) for detection of African swine fever virus (ASFV) in pigs and wild boars

Abstract

The study reports the development of a polymerase cross-linking spiral reaction (PCLSR) for the detection of African swine fever virus (ASFV) DNA in blood collected from infected pigs and wild boars. The method uses 3 specifically designed primers. Two outer-spiral primers comprising of 3′ sequences complementary to ASFV p72 gene sequence and 5′end sequences complementary to exogenous gene of black widow alpha-latrotoxin as well as additional ASFV specific cross-linking primer. The method is specific exclusively to ASFV DNA without cross-reactions with cDNA of classical swine fever virus (CSFV), porcine reproductive respiratory syndrome (PRRSV) or porcine epidemic diarrhea virus (PEDV). The sensitivity of this technique reached 7.2 × 102 copies per μl−1 of plasmid containing p72 gene. The PCLSR was conducted at 65 °C creating cross-linked complex structures. The results of PCLSR were visualized using SYBR Green I dye, gel electrophoresis while the reaction progress was traced using real-time PCR system that resulted in registration of fluorescent curves and melting peaks at 85.3 °C. The developed PCLSR was examined using blood or tissue samples collected from selected 17 ASF cases from infected wild boars and 3 outbreaks in pigs. Further tests have been also conducted using 55 tissue samples from 23 outbreaks and 22 cases. These results showed that PCLSR might be further used for preliminary and cost-effective detection and surveillance of ASFV.

Visual detection of porcine epidemic diarrhea virus using a novel reverse transcription polymerase spiral reaction method

“…Because of the difficulties in clinical diagnosis, the low accuracy of serological diagnosis, and the complicated operation of diagnosis via viral isolation methods, PSR technology has been widely used for the differential diagnosis of animal epidemics, such as canine parvovirus 2 (CPV-2) [18], African swine fever virus (ASFV) [19], and bovine herpesvirus 1 (BHV-1) [23]. PSR not only has the advantages of traditional PCR, as it can also detect pathogenic genes efficiently and specifically, it also has a shorter detection time, is easier to perform, and uses less reagents, when compared to traditional PCR.…”

“…Therefore, a pH-sensitive dye can be used to detect the product of the reaction with the naked eye [15]. PSR detection methods have been used for numerous human and veterinary pathogens [16][17][18][19]. Therefore, we evaluated clinical samples using the newly developed RT-PSR method to determine the method's utility for early detection of PEDV.…”

Simultaneous Nucleic Acids Detection and Elimination of Carryover Contamination With Nanoparticles-Based Biosensor- and Antarctic Thermal Sensitive Uracil-DNA-Glycosylase-Supplemented Polymerase Spiral Reaction

“…Particularly, indicating PSR results is a pivotal concern as simplifying detection tools is a major concern. Traditional monitoring techniques, including agarose gel electrophoresis, turbidimetry and visual detection, are not specific to target PSR amplicons; thus, they did not differentiate specific amplification from non-specific amplification (Wozniakowski et al, 2017). Moreover, these detection methods used for PSR assay required an additional analysis step (gel electrophoresis), optical instrument (i.e., turbidimeters), and special visual reagents (such as fluorescent), which restrict its wider application in various fields, such as point-of-care testing, "on-site" detection, and field diagnosis.…”

Rift Valley Fever Virus, Japanese Encephalitis Virus, and African Swine Fever Virus: Three Transboundary, Vector-Borne, Veterinary Biothreats With Diverse Surveillance, and Response Capacity Needs

“…Wozniakowski et al recently developed a polymerase cross-linking spiral reaction (PCLSR). This sensitive new test does not cross-react with other porcine pathogens, and has been validated on multiple sample types derived from ASFV-infected wild boars and pigs (115).…”