This study was conducted to obtain basic information for the use of the ATP fluorescence detection method in consideration of the most common and frequent contamination situation that occurs in laboratories dealing with fire blight causing bacterium, Erwinia amylovora. ATP luminescence measurements (Relative Light Unit, RLU) were tested against these pathogen cells (CFU/cm2) which were artificially introduced on the disinfected surface of a bench floor of a biosafety cabinet (Class 2 Type A1), on a part of the disinfected surface of a lab experimental bench, on a part of the disinfected floor, and on a part of the disinfected floor of an acryl chamber for bioaerosol studies in a biosafety laboratory (BSL 2 class) using two different ATP bioluminometers. RLU values were not much increased with the bacterial cells from 2.15 × 102/cm2 to 2.15 × 106/cm2. RLU values varied among the four different surfaces tested. RLU values measured from the same number of bacterial cells differed little between the two different ATP bioluminometers used for this study. RLU values obtained from bacterial cells higher than 2.15 × 107/cm2 indicated the presence of bacterial contamination on the four different surfaces tested. The R2 values obtained based on the correlation data for the RLU values in response to different E. amylovora cell numbers (CFU/ cm2) on the surfaces of the four test spots ranged from 0.9827 to 0.9999.

Recently, the importance of on-site detection of pathogens has drawn attention in the field of molecular diagnostics. Unlike in a laboratory environment, on-site detection of pathogens is performed under limited resources. In this study, we tried to optimize the experimental conditions for on-site detection of pathogens using a combination of ultra-fast convection polymerase chain reaction (cPCR), which does not require regular electricity, and nucleic acid lateral flow (NALF) immunoassay. Salmonella species was used as the model pathogen. DNA was amplified within 21 minutes (equivalent to 30 cycles of polymerase chain reaction) using ultra-fast cPCR, and the amplified DNA was detected within approximately 5 minutes using NALF immunoassay with nucleic acid detection (NAD) cassettes. In order to avoid false-positive results with NAD cassettes, we reduced the primer concentration or ultra-fast cPCR run time. For singleplex ultra-fast cPCR, the primer concentration needed to be lowered to 3 μM or the run time needed to be reduced to 14 minutes. For duplex ultra-fast cPCR, 2 μM of each primer set needed to be used or the run time needed to be reduced to 14 minutes. Under the conditions optimized in this study, the combination of ultra-fast cPCR and NALF immunoassay can be applied to on-site detection of pathogens. The combination can be easily applied to the detection of oral pathogens.

우리나라는 1996년 WTO/SPS(동식물검역협정)에 따라 검역병해충제도를 정비하였으며, 해외병해충의 위험으로부 터 자국내 생물자원과 자연환경을 보호하기 위하여 다양한 노력을 하고 있다. 현재까지의 수입식물 검역과정에서 검출된 병해충을 분석한 결과 병해충 검출건수는 수입량이 늘어나면서 증가하는 추세이다(00년 6,233건 → 17년 12,749건). 하지만 식물의 수입 유형에 따라 곡류, 사료류 등 비재식용 식물의 경우 병해충 검출률은 07년 이후 감소하고 있으며(07년 9.8% → 17년 3.3%), 묘목류, 종자류 등 재식용 식물의 경우 11년 이후로 지속적으로 증가하고 있어(11년 7.9% → 17년 22.0%) 재식용 식물을 통한 해외 병해충의 유입 위험도가 가증되고 있는 상황이다. 따라서 우리는 검역본부에서 운영하고 있는 자체 전산시스템인 병해충정보시스템(PIS)을 이용하여 2000년 이후 식물검역과 정에서 발견된 병해충 검출동향과 수입 유형에 따른 검출동향을 비교분석하고, 2016년 대비 2017년 병해충 검출동향을 파악하였다.

The purpose of this multiplex PCR assay is establishment and application for rapid and simultaneous detection of six pathogens related with insect diseases. Five pathogens were chosen based on the insect disease incidence rate in South Korea and specific primers of those pathogen were designed to detect insect diseases and test multiplex PCR for detecting Fungi; Beauveria bassiana(Bb), Metarhizium anisopliae(Ma), Bacteira; Bacillus thuringiensis(Bt), Pseudomonas aeruginosa(Pa), and Serratia marcescens(Sm). This research carried out the results detecting five kinds of insect pathogen of P. b. seulensis by multiplex PCR. Multiplex PCR is effective and save time to detect simultaneously these insect pathogens and multiple infections to prevent insect disease. In our study, using multiplex PCR, we demonstrated that P. b. seulensis was frequently infected with S. marcescens and co-infected with M. anisopliae in more than 80% of cases, indicating that such an analysis can be useful for pathogen identification, especially if different pathogens produce similar symptoms.