• Journal of Dairy Science and Biotechnology
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• 제33권3호
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• pp.197-202
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• 2015

While various foodborne pathogenic bacteria can be detected more rapidly via polymerase chain reaction than via conventional plating methods, it is impossible to distinguish between viable and dead cells in DNA-based assays. Hence, propidium monoazide (PMA) treatment has been introduced to detect living cells. The purpose of this study is to evaluate the applicability of the PMA treatment and real-time qPCR method for the detection of Cronobacter sakazakii and to compare it to that of plate counting. Based on our positive results, we suggest the use of PMA treatment and real-time qPCR for the detection of viable Cronobacter sakazakii in various food sources and an update of the Korean Food Code.

• 농업과학연구
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• 제49권4호
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• pp.795-807
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• 2022

The development and commercialization of industrial genetically modified (GM) organisms is actively progressing worldwide, highlighting an increased need for improved safety management protocols. We sought to establish an environmental monitoring method, using real-time polymerase chain reaction (PCR) and propidium monoazide (PMA) treatment to develop a quantitative detection protocol for living GM microorganisms. We developed a duplex TaqMan quantitative PCR (qPCR) assay to simultaneously detect the selectable antibiotic gene, ampicillin (AmpR), and the single-copy Escherichia coli taxon-specific gene, D-1-deoxyxylulose 5-phosphate synthase (dxs), using a direct cell suspension culture. We identified viable engineered E. coli cells by performing qPCR on PMA-treated cells. The theoretical cell density (true copy numbers) calculated from mean quantification cycle (Cq) values of PMA-qPCR showed a bias of 7.71% from the colony-forming unit (CFU), which was within ±25% of the acceptance criteria of the European Network of GMO Laboratories (ENGL). PMA-qPCR to detect AmpR and dxs was highly sensitive and was able to detect target genes from a 10,000-fold (10^-4) diluted cell suspension, with a limit of detection at 95% confidence (LOD_95%) of 134 viable E. coli cells. Compared to DNA-based qPCR methods, the cell suspension direct PMA-qPCR analysis provides reliable results and is a quick and accurate method to monitor living GM E. coli cells that can potentially be released into the environment.

• Restorative Dentistry and Endodontics
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• 제33권6호
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• pp.537-544
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• 2008

세균의 검출에 있어서 polymerase chain reaction (PCR) 방법은 기존의 plate counting과 달리 빠르게 세균을 검출할 수 있다. 하지만 세균이 죽은 후에도 DNA는 장기간 존재할 수 있기 때문에, DNA에 기초한 분석은 살아있는 세균과 죽은 세균을 구분할 수 없다. 최근에 DNA extraction전에 propidium monoazide (PMA)를 처리하여 살아있는 세균만 선택적으로 검출하는 방법이 제시되었다. PMA는 손상된 세포막만 통과하여 죽은 세포의 DNA와 빛 노출 하에서 결합하여 PCR이 증폭되는 것을 막는다. Enterococcus faecalis는 근관치료의 실패에 있어서 중요한 원인이 되는 세균으로 제시되어 왔다. 그리고 chlorhexidine (CHX)은 E. faecalis의 제거에 있어서 효과적인 약제임이 밝혀졌다. 이번 실험의 목적은 세균 수의 측정에 있어서, PMA 처리와 real-time PCR 방법의 적용 가능성을 기존의 plate counting과 비교하여 알아보는 것이다. 또한 E. faecalis에 대한 2% CHX의 살균 효과를 PMA 처리 후 real-time PCR 방법을 사용하여 알아보는 것이다. 실험 방법으로 먼저 살아있는 세균과 죽은 세균을 다른 비율로 섞어서 PMA를 처리한 후 real-time PCR을 시행하여 PMA가 빛 노출 하에서 죽은 세균의 DNA와 결합하는 효과를 나타내는지 알아보았다. 다음으로 PMA 처리 후 realtime PCR 방법을 이용하여 살아있는 세균의 양을 측정한 것을 plate counting으로 얻은 CFU와 비교하였다. 마지막으로 2% CHX의 처리시간을 다르게 하였을 때 E. faecalis에 대한 살균 효과를 PMA 처리 후 real-time PCR 방법을 사용하여 알아보았다. 실험 결과로 살아있는 E. faecalis의 비율이 감소할수록 Ct value는 증가하였다. 그리고 PMA 처리 후 real-time PCR 방법을 이용하여 세균의 양을 측정한 것과 plate counting으로 얻은 CFU 사이에는 Optical density (OD) 값이 1.0일 때까지는 상관관계가 있었다. 하지만 OD 값이 1.5일 때는, PMA를 처리한 후 real-time PCR을 시행했을 때 측정된 살아있는 세균의 양이 감소하였음에 반해서 plate counting에 의한 CFU는 계속 증가하였다. 마지막으로 2% CHX을 오래 적용할수록 살아있는 E. faecalis의 상대적인 양이 감소하는 것을 PMA 처리와 real-time PCR 방법을 이용해 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제23권12호
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• pp.1708-1716
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• 2013

Conventional molecular detection methods cannot distinguish between viable and dead Escherichia coli O157 cells. In this study, the loop-mediated isothermal amplification (LAMP) method combined with propidium monoazide (PMA) treatment was developed to selectively detect viable E. coli O157 cells. Four primers, including outer primers and inner primers, were specially designed for the recognition of six distinct sequences on the serogroups (O157) of the specific rfbE gene of the E. coli O157 genome. PMA selectively penetrated through the compromised cell membranes and intercalated into DNA. Amplification of DNA from dead cells was completely inhibited by $3.0{\mu}g/ml$ PMA, whereas the DNA derived from viable cells was amplified remarkably within 1 h by PMA-LAMP. Exhibiting high sensitivity and specificity, PMA-LAMP is a suitable method for evaluating the inactivation efficacy of slightly acidic electrolyzed water in broth. PMA-LAMP can selectively detect viable E. coli O157 cells. This study offers a novel molecular detection method to distinguish between viable and dead E. coli O157 cells.

• 한국어병학회지
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• 제37권1호
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• pp.49-60
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• 2024

A viable but non-culturable (VBNC) state is a survival strategy adopted by bacteria when faced with unfavorable environmental conditions, rendering them unable to grow on nutrient agar while maintaining low metabolic activity. This study explored the impact of temperature and nutrient availability on inducing VBNC state in Edwardsiella piscicida, the most important bacterial fish pathogen, and assessed its pathogenicity at VBNC state. E. piscicida was suspended in filtered sterile seawater and exposed to three different temperatures (4, 10, and 25℃) to induce the VBNC state. Subsequently, the induced VBNC cells were subjected to resuscitation by either raising the temperature to 28℃ or inoculating them in brain heart infusion broth supplemented with 1% NaCl. A propidium monoazide (PMA)-qPCR method was also developed to selectively quantify live (VBNC or culturable) E. piscicida cells. The results showed that the bacteria entered the VBNC state after approximately 1 month at 4℃ and 25℃, and 2 months at 10℃. The VBNC E. piscicida cells were successfully revived within 3 days in a nutrient-rich environment at 28℃, highlighting the significance of temperature and nutrition in inducing and resuscitating the VBNC state. In pathogenicity tests, resuscitated E. piscicida cells exhibited high pathogenicity in olive flounder comparable to cultured bacteria, while VBNC cells showed no signs of infection, suggesting they are unlikely to resuscitate in fish. In conclusion, this study contributes to our understanding of fish pathogen ecology by investigating the characteristics of the VBNC state under varying temperature and nutrition conditions.

• Journal of Microbiology and Biotechnology
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• 제25권6호
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• pp.863-871
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• 2015

Bifidobacterium longum subsp. longum BBMN68, isolated from centenarians in Guangxi, China, has been proved to be a promising probiotic strain for its health benefits. In this study, the biodistribution of this strain in the rat gut was first investigated using the quantitative realtime PCR assay and propidium monoazide. Strain-specific primers were originally designed based on the BBMN68 genome sequence. Healthy rats were orally inoculated with either a single dose of BBMN68 (10¹⁰ colony-forming units/kg), or with one dose per day for 7 days and bacterial concentrations were analyzed in detail from the intestinal contents and feces of four different gut locations, including stomach, small intestine, colon, and rectum. Results indicated that strain BBMN68 could overcome the rigors of passage through the upper gastrointestinal tract and transiently accumulate in the colon, even though survival in the stomach and small intestine was not high. A good level of BBMN8 could stay in vivo for 72 h following a 7-day oral administration, and a daily administration is suggested for a considerable and continuous population of BBMN68 to be maintained in the host intestine.

• Journal of Microbiology and Biotechnology
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• 제27권5호
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• pp.995-1004
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• 2017

Culture-dependent methods, such as heterotrophic plate counting (HPC), are usually applied to evaluate the bacteriological quality of hemodialysis water. However, these methods cannot detect the uncultured or viable but non-culturable (VBNC) bacteria, both of which may be quantitatively predominant throughout the hemodialysis water treatment system. Therefore, propidium monoazide (PMA)-qPCR associated with HPC was used together to profile the distribution of the total viable bacteria in such a system. Moreover, high-throughput sequencing of 16S rRNA gene amplicons was utilized to analyze the microbial community structure and diversity. The HPC results indicated that the total bacterial counts conformed to the standards, yet the bacteria amounts were abruptly enhanced after carbon filter treatment. Nevertheless, the bacterial counts detected by PMA-qPCR, with the highest levels of $2.14{\times}10^7copies/100ml$ in softener water, were much higher than the corresponding HPC results, which demonstrated the occurrence of numerous uncultured or VBNC bacteria among the entire system before reverse osmosis (RO). In addition, the microbial community structure was very different and the diversity was enhanced after the carbon filter. Although the diversity was minimized after RO treatment, pathogens such as Escherichia could still be detected in the RO effluent. In general, both the amounts of bacteria and the complexity of microbial community in the hemodialysis water treatment system revealed by molecular approaches were much higher than by traditional method. These results suggested the higher health risk potential for hemodialysis patients from the up-to-standard water. The treatment process could also be optimized, based on the results of this study.

• 한국수산과학회지
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• 제54권1호
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• pp.16-22
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• 2021

This study investigated the reduction in human norovirus (HNV) GII. 4 count in pacific oyster Crassostrea gigas using electron beam irradiation. Infectious HNV GII. 4 was detected using RT-qPCR (real time reverse transcription-quantitative polymerase chain reaction) with PMA (propidium monoazide)/sarkosyl. At electron beam doses 1, 5, 7, and 10 kGy, the count of HNV GII. 4 was 2.74, 2.37, 2.06, and 1.55 log copies/μL (control, 3.01 log copy/μL), respectively, confirming that as the irradiation dose increased, norovirus count reduced significantly (P<0.05). After PMA/sarkosyl treatment, the counts further reduced at the same irradiation dose, and 10 kGy showed significant differences between the non-treated and PMA/sarkosyl-treated samples (P<0.05). The Ed (decimal reduction dose of electron beam) value based on the first-order kinetic model was 7.33 kGy (R2=0.98). No significant difference was observed in the pH values of the control (6.2) and electron beam-irradiated samples at all doses (6.1). For sensory evaluation, the non-treated sample scored the highest in all categories (5.25-6.17), while the samples treated with 10 kGy showed the lowest score (4.67-5.33), although without statistical significance (P>0.05). Overall, our results suggest that 7 kGy electron beam is sufficient for the non-thermal sterilization of oysters without causing significant changes in quality.