구제역바이러스 혈청형 A 검출을 위한 peptide nucleic acid (PNA)기반 multiplex real-time RT-PCR 개발

Developing peptide nucleic acid based multiplex real time RT-PCR to detect Foot-and-Mouth-Disease virus Serotype A

Abstract

There have been a total tenth FMD outbreaks in Korea and for the first time, type O and A were detected simultaneously in 2017, which led to difficulties in FMD control. For the effective prevention of FMD, the importance of discrimination of serotypes became greater. Therefore, the most urgent requirement in case of FMD outbreak is differential diagnosis of serotypes. In this study, we developed a PNA probe-mediated multiplex real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay using the peptide nucleic acid (PNA) probe, which is known to be stable to nucleotide mutation and that could specifically detect the all FMDV serotype A, FMDVA Yeoncheon strain which was occurred in Korea in 2017, and FMDV A viruses shown 96% similarity with FMDVA/Yeoncheon strain, at the same time. Therefore, It is believed that the newly introduced FMDVA will be effectively diagnosed using the PNA probe multiplex RT-PCR developed in this study, and ultimately contribute to the prevention of FMD.

Fig. 1. Results of PNA probe mediated real-time reverse transcription-polymerase chain reaction(rRT-PCR) using 9 FMDV A strains. All strains (n=9) of FMDV A were identified in the hexachloro fluorescein (HEX) channel at 49~66°C. The Yeoncheon strain (I) was identified in the FAM channel at 66°C. The A/Yeoncheon 96% similarity strains (A, B, C, F, G, H and I) were identified in the TxR channel at 60~73°C. Moreover, FMDV A/Iran-05 and FMDV A/A22Iraq which were different genotype with FMDVA/ASIA/SEA97 were only identified in HEX channel at 49°C and 64°C respectively. (A) FMDVA/Pocheon, (B) FMDV A/Vietnam (VN15), (C) FMDVA/Malaysia97, (D) FMDVA/Iran05, (E) FMDVA/A22 Iraq, (F) FMDVA/zabaykalsky, (G) FMDVA/Vietnam(VN/2013), (H) FMDVA/Vietnam (VN18), (I) FMDV A/Yeoncheon.

Fig. 2. Results of PNA probe mediated rRT-PCR using the total of 32 strains (FMDV (n=31) and SVV (n=1). The six FMDV serotypes (FMDVO (n=15), FMDVAsia1 (n=3), FMDVC (n=1), FMDVSAT1 (n=1), FMDVSAT2 (n=1), FMDVSAT3 (n=1)) and Seneca Valley virus (n=1) were used to assess the specificity of the PNA mediated rRT-PCR. There were no melting curve peaks in all 32 viruses. (A) FMDVO(n=15), (B) FMDV Asia1 (n=3), (C) FMDVC (n=1), (D) SAT1 (n=1), (E) SAT2 (n=1), (F) SAT3 (n=1), (G) Seneca valley virus (SVV) (n=1) and (H) Non-template control (NTC).

Table 1. Sequence of primers and probes used in rRT-PCR

Table 2. The list of Foot-and-mouth disease virus (FMDV) and Seneca vally virus (SVV) used in this experiment