The detection of Mycobacterium bovis (M. bovis) in environmental samples with precision is imperative to control bovine tuberculosis (bTB) infections at the herd level, as residual M. bovis remains one of the major causes of recurring infections. In this study, a nested PCR method for the detection of M. bovis in environmental samples was applied to identify potential environmental reservoirs of the bacterium. A set of 200 environmental samples (167 fecal samples and 33 water samples) from 39 herds with a history of bTB outbreak was analyzed using a nested PCR method to detect residual M. bovis. Amplicon libraries of the IS6110 target gene fragment were amplified from M. bovis DNA using two established primer sets. A positive nested PCR result was observed in 69.5% of fecal samples and 66.7% of water samples, thus showing that residual M. bovis was present in the environmental samples of bTB-positive herds in a high proportion. This study is the first to demonstrate high levels of M. bovis DNA in environmental samples and to show that environmental reservoirs of this pathogen contribute to recurring outbreaks of bTB. Environmental monitoring of herds in which bTB outbreaks have occurred with high sensitivity and specificity is expected to help prevent the recurrence of potential bTB disease and improve the herd environment.

Pelargonium zonate spot virus (PZSV)는 group IV (+) ssRNA viruses, Bromoviridae에 속하는 식물 병원체로, 일반적으로 토마토, 국화, 아티초크 및 제라늄에 감염된다. 본 연구는 검역 현장에서 PZSV를 신속하고 특이적으로 진단 할 수 있는 PCR module을 개발하는 것을 목적으로 하였다. PZSV를 검출하기 위한 RT-PCR 프라이머 선발 결과, 각각 513 및 320 bp를 증폭하는2개 조합을 선발하으며, 더욱 높은 검출감도로 검출할 수 있을 뿐아니라 RT-PCR을 검증할 수 있는 nested PCR 프라이머 조합을 개발하였다. 또한, 제한효소 Xho I 부위를 삽입한 유전자변형-양성대조구 플라스미드를 설계하여, PCR module에서 대조구로부터 오염을 검증할 수 있도록 개발하였다. 본 연구에서 개발한 PCR module은 토마토, 국화, 아티초크 및 제라늄 등에서 PZSV를 간편, 신속 및 특이적으로 검출하여, 지속적으로 식물검역에 활용할 수 있을 것으로 기대된다.

종자의 수입 시, 검역관련 종자전염바이러스는 가장 문제가 되는 식물병이다. 본 연구에서 PCR 검역체계가 보고되지 않은 3종의 종자전염바이러스, Cherry rasp leaf virus (CRLV), Spinach latent virus (SpLV) 및 White clover mosaic virus (WClMV)를 검출하기 위하여 reverse transcription polymerase chain reaction (RT-PCR)과 nested polymerase chain reaction (nested PCR) 방법을 도입하였다. 각각의 바이러스별로 2 세트의 RT-PCR primer가 선발되었으며, 증폭산물에서 더욱 높은 감도로 검출 할 수 있는 nested PCR primer set를 개발하였다. 본 연구에서 사용한 RT-PCR과 nested PCR 방법은 종자로부터 CRLV, SpLV 및 WClMV를 검역하는 고효율적 진단시스템으로 제공될 것이다.

Chrysanthemum stunt viroid (CSVd) is a serious pathogen affecting chrysanthemum that has caused significant economic losses to Chrysanthemum flower production worldwide. Control of CSVd disease is difficult due to its contagious nature and long latent period in the field. As chrysanthemum is most often produced by implanting seedlings, it is necessary to diagnose CSVd infection before cultivation. In this study, we screened CSVd infection in seedlings from 30 varieties including 5 domestic, 6 Japanese, and 19 European varieties. Molecular diagnosis of the combination of RT-PCR and nested PCR showed that CSVd was not detected by the first RT-PCR but detected by the second nested PCR analysis in 10 varieties, including 1 domestic, 2 Japanese, and 7 European varieties. Further comparison of 10 identified CSVd nucleotide sequences showed that those are highly conserved (99-100%) and the most similar to an isolate (AB006737) identified in Hokkaido, Japan. Our study suggests that the combination of RT-PCR and nested PCR analysis is successful for the CSVd diagnosis of seedlings and the molecular diagnosis is necessary to prevent the introduction and propagation of viroid disease into the fields.