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.

The bumblebee, Bombus terrestris, plays an important role as one of alternative pollinators since the outbreak of honeybee colony collapse disorder. Recently, pathogens and parasites such as viruses, bacteria and mites affecting the life span and fecundity of their host have been discovered in B. terrestris. In this study, in order to detect viral infection in B. terrestris, we collected B. terrestris adults and isolated total RNA for diagnostic PCR. The PCR primers specific for pathogenic viruses were newly designed and applied to gene amplification for cloning and detection. Capsid protein gene of black queen cell virus (BQCV) among examined viral genes was only successfully amplified from collected bumble bee adults and sequenced. To optimize the detection of capsid protein gene of BQCV, 4 regions in the capsid protein gene were selected and further analyzed in quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis revealed that capsid protein gene was directly detected with not more than 200 ng total RNA. This result suggests that an optimized detection via qRT-PCR can be applied for the rapid and sensitive diagnosis of BQCV infection in the field population as well as risk assessment of B. terrestris.

A rapid and sensitive assay for specific detection and identification of barley yellow mosaic virus(BaYMV) was set up using the reverse transcriptase polymerase chain reaction(RT-PCR). A couple of primers was select to discriminate the viruses. PCR fragments of BaYMV(ca.0.9 kb) were obtained by using the method designed for BaYMV capsid protein. RT-PCR fragments were cloned with vector pT7 Blue and the resulting clones were sequenced. Capsid protein of BaYMV consisted of 297 amino acids and 891 nucleotides. The capsid protein sequence of BaYMV showed that 98% of nucleotides and 99% of amino acids homology.