Development of a ladder-shape melting temperature isothermal amplification (LMTIA) assay for detection of African swine fever virus (ASFV)

Abstract

Background Due to the unavailability of an effective vaccine or antiviral drug against the African swine fever virus (ASFV), rapid diagnosis methods are needed to prevent highly contagious African swine fever. Objectives The objective of this study was to establish the ladder-shape melting temperature isothermal amplification (LMTIA) assay for the detection of ASFV. Methods LMTIA primers were designed with the p72 gene of ASFV as the target, and plasmid pUC57 was used to clone the gene. The LMTIA reaction system was optimized with the plasmid as the positive control, and the performance of the LMTIA assay was compared with that of the commercial real-time polymerase chain reaction (PCR) kit in terms of sensitivity and detection rate using 200 serum samples. Results Our results showed that the LMTIA assay could detect the 10 4 dilution of DNA extracted from the positive reference serum sample, which was the same as that of the commercial real-time PCR kit. The coincidence rate between the two assays was 100%. Conclusions The LMTIA assay had high sensitivity, good detection, and simple operation. Thus, it is suitable for facilitating preliminary and cost-effective surveillance for the prevention and control of ASFV.

Rapid Visible Detection of African Swine Fever Virus Using Hybridization Chain Reaction‐Sensitized Magnetic Nanoclusters and Affinity Chromatography

“…[ 30,47 ] The DNA fragments were obtained by digestion with EcoRI and used directly for the assay. [ 48 ] PAGE analysis confirmed that the prepared DNA fragments for each target gene hybridized with the corresponding probe DNA, and the amplified products were obtained after performing HCR (Figure S11a,b, Supporting Information). Additionally, the probe and helper DNA specific to the B646L gene generated amplicons only for the B646L gene target, implying that our HCR hairpins were highly specific to each target (Figure S11c, Supporting Information).…”

Current detection methods of African swine fever virus

“…Wang et al has devised a ladder-shaped melting temperature isothermal amplification (LMTIA) technique that specifically targets the ASFV-B646L gene. This method exhibits the same sensitivity of commercially available qPCR reagent kits with a LOD of 0.5 copies/μL (119). Furthermore, the integration of LAMP with carbon nanodots (CND) technology has facilitated the development of a fluorescent biosensor, enabling the highly sensitive detection of ASFV with a detection sensitivity of 15.21 copies/μL (65).…”

Rapid Discrimination of Panax quinquefolium and Panax ginseng Using the Proofman-Duplex-LMTIA Technique

“…Compared to traditional PCR, LMTIA offers several advantages: (1) it does not rely on expensive PCR or qPCR instruments, employing isothermal amplification; (2) rapid reaction, with each reaction taking only 30 min; (3) suitable for shorter target sequences, requiring a minimum length of 60 bp for amplification; (4) high sensitivity, with studies showing 50 times higher sensitivity compared to Loop-mediated isothermal amplification (LAMP) technology [9]; and (5) broad applicability, as the employed Bst enzyme possesses reverse transcriptase activity, making it suitable for various nucleic acid sequences, including RNA. This technique has been successfully applied in diverse fields, such as the rapid detection of cassava components in sweet potato starch [10] and African swine fever virus detection [11].…”