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. 2022 Jul;23(4):e51.
doi: 10.4142/jvs.22001. Epub 2022 May 2.

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

Yongzhen Wang #  1 , Borui Wang #  2 , Dandan Xu  2 , Meng Zhang  2 , Xiaohua Zhang  1 , Deguo Wang  3
Affiliations

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

Yongzhen Wang et al. J Vet Sci. 2022 Jul.

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 104 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.

Keywords: African swine fever (ASF); African swine fever virus (ASFV); commercial real-time PCR kit; ladder-shape melting temperature isothermal amplification (LMTIA).

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig. 1
Fig. 1. Ladder-type melting temperature curve of target sequences.
Fig. 2
Fig. 2. Restriction digestion map of pUC57 plasmid containing 376 nt fragment of ASFV p72 gene. Note: Lane 1: Plasmid; Lane 2: Plasmid digested with EcoRI-SalI; Lane M: 1 kb DNA Marker.
Fig. 3
Fig. 3. Amplification plot of the LMTIA reaction at 54°C, 55°C, and 56°C.
LMTIA, ladder-shape melting temperature isothermal amplification; RFU, relative fluorescence units.
Fig. 4
Fig. 4. Sensitivity of the developed LMTIA assay with pUC57-p72 DNA as template.
Note: A: 1 pg pUC57-p72 DNA; B: 100 fg pUC57-p72 DNA; C: 10 fg pUC57-p72 DNA; D: 1 fg pUC57-p72 DNA; E: 0.1 fg pUC57-p72 DNA; F: 0.01 fg pUC57-p72; G: ddH2O. LMTIA, ladder-shape melting temperature isothermal amplification; RFU, relative fluorescence units.
Fig. 5
Fig. 5. Sensitivity of the developed LMTIA assay for detection of the genomic DNA of ASFV.
Note: A: Positive controls (1 pg pUC57-p72 DNA); B: 10−1 dillution of genomic DNA extracted from ASFV-positive serum sample; C: 10−2 dillution of genomic DNA extracted from ASFV-positive serum sample; D: 10−3 dillution of genomic DNA extracted from ASFV-positive serum sample; E: 10−4 dillution of genomic DNA extracted from ASFV-positive serum sample; F: 10−5 dillution of genomic DNA extracted from ASFV-positive serum sample; G: ddH2O. LMTIA, ladder-shape melting temperature isothermal amplification; ASFV, African swine fever virus; RFU, relative fluorescence units.
Fig. 6
Fig. 6. Sensitivity of the commercial Real-time PCR Kit for detection of the genomic DNA of ASFV.
Note: A: Positive controls (1 pg pUC57-p72 DNA); B: 10−1 dillution of genomic DNA extracted from ASFV-positive serum sample; C: 10−2 dillution of genomic DNA extracted from ASFV-positive serum sample; D: 10−3 dillution of genomic DNA extracted from ASFV-positive serum sample; E: 10−4 dillution of genomic DNA extracted from ASFV-positive serum sample; F: 10−5 dillution of genomic DNA extracted from ASFV-positive serum sample; G: ddH2O. PCR, polymerase chain reaction; ASFV, African swine fever virus; RFU, relative fluorescence units.
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References

    1. Njau EP, Machuka EM, Cleaveland S, Shirima GM, Kusiluka LJ, Okoth EA, et al. African swine fever virus (ASFV): biology, genomics and genotypes circulating in sub-Saharan Africa. Viruses. 2021;13(11):2285. - PMC - PubMed
    1. Pikalo J, Deutschmann P, Fischer M, Roszyk H, Beer M, Blome S. African swine fever laboratory diagnosis-lessons learned from recent animal trials. Pathogens. 2021;10(2):177. - PMC - PubMed
    1. Yuan F, Petrovan V, Gimenez-Lirola LG, Zimmerman JJ, Rowland RR, Fang Y. Development of a blocking enzyme-linked immunosorbent assay for detection of antibodies against African swine fever virus. Pathogens. 2021;10(6):760. - PMC - PubMed
    1. Zhang X, Liu X, Wu X, Ren W, Zou Y, Xia X, et al. A colloidal gold test strip assay for the detection of African swine fever virus based on two monoclonal antibodies against P30. Arch Virol. 2021;166(3):871–879. - PubMed
    1. Chen C, Lai H, Liang H, He Y, Guo G, Li L. A new method for detection African swine fever virus: time-resolved fluorescence immunoassay. J Fluoresc. 2021;31(5):1291–1296. - PMC - PubMed