African Swine Fever

A comparison of real-time PCR (rPCR), loop-mediated isothermal amplification (LAMP), and antigen ELISA were compared as leading tests for ASFV identification. rPCR is the most studied and regularly used method but requires a thermocycler. LAMP is a more recently developed method and must be optimized, particularly for determining a cut-off time. However, it shows comparable sensitivity to rPCR, especially for well preserved samples. For samples that had undergone freeze-thaw, time required for amplification varied widely for LAMP suggesting controlled sample storage is more important for LAMP. However, LAMP also has advantages over rPCR in that it is isothermal and less sensitive to contaminants that cause false negatives in PCR. Antigen ELISA is overall less sensitive, especially in poorly preserved samples. However, ELISA may be useful to confirm other assays and as a herd test. ELISA does have the advantage of detecting viral antigen not DNA, lowering the likelihood of a false diagnosis from old viral DNA. PCR assays for ASFV in ticks (the vector of ASFV) are also in development though LAMP and ELISA have yet been tested.

Polymerase cross-linking spiral reaction (PCLSR) is a method of amplifying targeted regions of DNA and can be used as a low cost way to check for the presence of viral DNA. Wozniakowski, et al. amplified a region of the p72 gene from African Swine Fever Virus. The reaction is optimized when performed at 65℃ steadily for 45 minutes. The procedure requires GspSSD polymerase for amplification, SYBR Green® I to fluorescently label the double stranded DNA product, and 2 sets of modified primers (sequences specific to ASFV included in methods). The primers include 3 major regions: 1 set covers the target region, 1 set flanks the target region, and 1 set protects the DNA in early steps and initiates cross-linked spiraling at the end. The third set originated from an unrelated Latrodectus hesperus gene designed to bind to itself but not the region of ASFV DNA. The estimated cost of each reaction is 2€ ($2.36).

Fernández‐Pinero, J. , Gallardo, C. , Elizalde, M. , Robles, A. , Gómez, C. , Bishop, R. , Heath, L. , Couacy‐Hymann, E. , Fasina, F. O., Pelayo, V. , Soler, A. and Arias, M. 2013. Molecular Diagnosis of African Swine Fever by a New Real‐Time PCR Using Universal Probe Library.

A set of PCR primers for the ASFV capsid protein gene VP72 was used for real time PCR. The primers were specific enough for all of the purposes in this study and could likely be used for other variations of PCR for ASFV.

A recombinase polymerase amplification (RPA) reaction for the detection of ASFV was developed. The reaction is isothermal at 39℃ and takes only 20 minutes. The primers targeted the p72 gene which was specific enough to distinguish ASFV from other viruses that commonly infect pigs including Classical Swine Fever Virus and Foot-and-Mouth Disease Virus. RPA requires fewer primers than LAMP and is less likely to result in false positives. However, it uses fluorescence for detection. The authors used the portable Genie III Scanner which costs $18,000 to detect the product.

Biagetti, M., Cuccioloni, M., Bonfili, L., Cecarini, V., Sebastiani, C., Curcio, L., Giammarioli, M., De Mia, G.M., Eleuteri, A.M., Angeletti, M. 2018. [Chimeric DNA/LNA-based biosensor for the rapid detection of African swine fever virus.] (see Eric for full article)

ASFV can be detected using a chimeric DNA/LNA (locked nucleic acid) biosensor. DNA is extracted from infected blood samples and passed over the DNA/LNA complex bound by streptavidin to a cuvette. The sensor is designed to bind to a region of the p72 gene specific to ASFV. This process takes about 5 minutes. Binding can be read without additional fluorescent labels but must be read by an evanescent wave/resonant mirror biosensor. Sensors such as these are being improved but currently cost around $15,000. The biosensor DNA/LNA complex can be washed and reused approximately 40 times.

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