• 산업식품공학
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• 제21권3호
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• pp.191-200
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• 2017

There have been great efforts to develop a rapid and sensitive detection method to monitor the presence of pathogenic bacteria in food. While a number of methods have been reported for bacterial detection with a detection limit to a single digit, most of them are suitable only for the bacteria in pure culture or buffered solution. On the other hand, foods are composed of highly complicated matrices containing carbohydrate, fat, protein, fibers, and many other components whose composition varies from one food to the other. Furthermore, many components in food interfere with the downstream detection process, which significantly affect the sensitivity and selectivity of the detection. Therefore, isolating and concentrating the target pathogenic bacteria from food matrices are of importance to enhance the detection power of the system. The present review provides an introduction to the representative sample preparation strategies to isolate target pathogenic bacteria from food sample. We further describe the nucleic acid-based detection methods, such as PCR, real-time PCR, NASBA, RCA, LCR, and LAMP. Nucleic acid-based methods are by far the most sensitive and effective for the detection of a low number of target pathogens whose performance is greatly improved by combining with the sample preparation methods.

• The Plant Pathology Journal
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• 제35권2호
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• pp.164-171
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• 2019

An assay for detecting Apple scar skin viroid (ASSVd) was developed based on nucleic acid sequence based amplification (NASBA) in combination with realtime detection during the amplification process using molecular beacon. The ASSVd specific primers for amplification of the viroid RNA and molecular beacon for detecting the viroid were designed based on highly conserved regions of several ASSVd sequences including Korean isolate. The assay had a detection range of $1{\times}10^4$ to $1{\times}10^{12}$ ASSVd RNA $copies/{\mu}l$ with reproducibility and precision. Following the construction of standard curves based on time to positive (TTP) value for the serial dilutions ranging from $1{\times}10^7$ to $1{\times}10^{12}$ copies of the recombinant plasmid, a standard regression line was constructed by plotting the TTP values versus the logarithm of the starting ASSVd RNA copy number of 10-fold dilutions each. Compared to the established RT-PCR methods, our method was more sensitive for detecting ASSVd. The real-time quantitative NASBA method will be fast, sensitive, and reliable for routine diagnosis and selection of viroid-free stock materials. Furthermore, real-time quantitative NASBA may be especially useful for detecting low levels in apple trees with early viroid-infection stage and for monitoring the influence on tree growth.

• Journal of Microbiology and Biotechnology
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• 제24권3호
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• pp.297-312
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• 2014

Food safety is increasingly becoming an important public health issue, as foodborne diseases present a widespread and growing public health problem in both developed and developing countries. The rapid and precise monitoring and detection of foodborne pathogens are some of the most effective ways to control and prevent human foodborne infections. Traditional microbiological detection and identification methods for foodborne pathogens are well known to be time consuming and laborious as they are increasingly being perceived as insufficient to meet the demands of rapid food testing. Recently, various kinds of rapid detection, identification, and monitoring methods have been developed for foodborne pathogens, including nucleic-acid-based methods, immunological methods, and biosensor-based methods, etc. This article reviews the principles, characteristics, and applications of recent rapid detection methods for foodborne pathogens.

• International Journal of Oral Biology
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• 제46권1호
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• pp.1-6
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• 2021

Since the outbreak of coronavirus disease 2019 (COVID-2019), the infection has spread worldwide due to the highly contagious nature of severe acute syndrome coronavirus (SARS-CoV-2). To manage SARS-CoV-2, the development of diagnostic assays that can quickly and accurately identify the disease in patients is necessary. Currently, nucleic acid-based testing and serology-based testing are two widely used approaches. Of these, nucleic acid-based testing with quantitative reverse transcription-PCR (RT-qPCR) using nasopharyngeal (NP) and/or oropharyngeal (OP) swabs is considered to be the gold standard. Recently, the use of saliva samples has been considered as an alternative method of sample collection. Compared to the NP and OP swab methods, saliva specimens have several advantages. Saliva specimens are easier to collect. Self-collection of saliva specimens can reduce the risk of infection to healthcare providers and reduce sample collection time and cost. Until recently, the sensitivity and accuracy of the data obtained using saliva specimens for SARS-CoV-2 detection was controversial. However, recent clinical research has found that sensitive and reliable data can be obtained from saliva specimens using RT-qPCR, with approximately 81% to 95% correspondence with the data obtained from NP and OP swabs. These data suggest that self-collected saliva is an alternative option for the diagnosis of COVID-19.

• Gut and Liver
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• 제12권6호
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• pp.641-647
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• 2018

Background/Aims: Helicobacter pylori eradication rates are decreasing because of increases in clarithromycin resistance. Thus, finding an easy and accurate method of detecting clarithromycin resistance is important. Methods: We evaluated 70 H. pylori isolates from Korean patients. Dual-labeled peptide nucleic acid (PNA) probes were designed to detect resistance associated with point mutations in 23S ribosomal ribonucleic acid gene domain V (A2142G, A2143G, and T2182C). Data were analyzed by probe-based fluorescence melting curve analysis based on probe-target dissociation temperatures and compared with Sanger sequencing. Results: Among 70 H. pylori isolates, 0, 16, and 58 isolates contained A2142G, A2143G, and T2182C mutations, respectively. PNA probe-based analysis exhibited 100.0% positive predictive values for A2142G and A2143G and a 98.3% positive predictive value for T2182C. PNA probe-based analysis results correlated with 98.6% of Sanger sequencing results (${\kappa}$-value=0.990; standard error, 0.010). Conclusions: H. pylori clarithromycin resistance can be easily and accurately assessed by dual-labeled PNA probe-based melting curve analysis if probes are used based on the appropriate resistance-related mutations. This method is fast, simple, accurate, and adaptable for clinical samples. It may help clinicians choose a precise eradication regimen.

• 대한의용생체공학회:의공학회지
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• 제42권3호
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• pp.125-142
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• 2021

The POCT (point-of-care test) sensing that has been a fast-developing field is expected to be a next generation technology in health care. The POCT sensors for the detection of proteins, small molecules and especially nucleic acids have lately attracted considerable attention. According to the World Health Organization (WHO), the POCT methods are required to follow the ASSURED guidelines (Affordable, Sensitive, Specific, User- friendly, Robust and rapid, Equipment-free, Deliverable to all people who need the test). Recently, several CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based diagnostic techniques using the sensitive gene recognition function of CRISPR have been reported. CRISPR/Cas (Cas, CRISPR associated protein) systems based detection technology is the most innovative gene analysis technology that is following the ASSURED guidelines. It is being re-emerged as a powerful diagnostic tool that can detect nucleic acids due to its characteristics that enable rapid, sensitive and specific analyses of nucleic acid. The first CRISPR-based diagnosis begins with the discovery of the additional function of Cas13a. The enzymatic cleavage occurs when the conjugate of Cas protein and CRISPR RNA (crRNA) detect a specific complementary sequence of the target sequence. Enzymatic cleavage occurs on not only the target sequence, but also all surrounding non-target single-stranded RNAs. This discovery was immediately utilized as a biosensor, and numerous sensor studies using CRISPR have been reported since then. In this review, the concept of CRISPR, the characteristics of the Cas protein required for CRISPR diagnosis, the current research trends of CRISPR diagnostic technology, and some aspects to be improved in the future are covered.

• 한국산학기술학회논문지
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• 제20권12호
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• pp.117-124
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• 2019

비브리오 콜레라는 수산물과 선박평형수 내에서 모니터링되고 있는 중요 병원성 박테리아이다. 이를 검출하기 위한 여러 방법들이 개발되어 왔으나, 시간 소모가 크고 민감도에서 한계가 있었다. 본 연구는 비브리오 콜레라를 보다 정확하게 검출하기 위한 방법을 개발하는 목적으로 수행하였다. PNA 기반 비대칭 real-time PCR 기술에 적용하기 위하여 펩티드 핵산(Peptide nucleic acid, PNA) 프로브를 개발하였다. 독성 유전자인 Cholera enterotoxin subunit B (ctxB)를 비브리오 콜레라 검출을 위한 타겟 유전자로 선정하고, conventional PCR과 real-time PCR을 위한 positive template를 합성하였다. Real-time PCR 프라이머와 PNA 프로브를 디자인하여, 정량 분석을 위한 표준곡선 실험을 수행하였다. 선택된 PNA 프로브는 비브리오 콜레라에 특이적으로 반응하였으며, 검출한계는 0.1 cfu/100 mL이었다. 종합해 보면, 본 연구에서 개발된 PNA 프로브와 비대칭 real-time PCR 방법은 수산물과 선박평형수 뿐만 아니라 해양환경에 있는 비브리오 콜레라를 신속하고 정확하게 모니터링할 수 있는 기술로 판단된다.

• 한국해양바이오학회지
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• 제6권1호
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• pp.1-7
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• 2014

Marine-derived pathogens threat health and life of human and animals. Therefore, rapid and specific detection methods need to be developed. Here, we summarized various groups of detection methods, including conventional method, flow cytometry, nucleic acid-based method, and protein-based method. In addition, perspective of detection technique was discussed as a unified detection system for pathogens.

• Asian-Australasian Journal of Animal Sciences
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• 제20권2호
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• pp.283-294
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• 2007

Conventional culture-based methods of enumerating rumen microorganisms (bacteria, archaea, protozoa, and fungi) are being rapidly replaced by nucleic acid-based techniques which can be used to characterise complex microbial communities without incubation. The foundation of these techniques is 16S/18S rDNA sequence analysis which has provided a phylogenetically based classification scheme for enumeration and identification of microbial community members. While these analyses are very informative for determining the composition of the microbial community and monitoring changes in population size, they can only infer function based on these observations. The next step in functional analysis of the ecosystem is to measure how specific and, or, predominant members of the ecosystem are operating and interacting with other groups. It is also apparent that techniques which optimise the analysis of complex microbial communities rather than the detection of single organisms will need to address the issues of high throughput analysis using many primers/probes in a single sample. Nearly all the molecular ecological techniques are dependant upon the efficient extraction of high quality DNA/RNA representing the diversity of ruminal microbial communities. Recent reviews and technical manuals written on the subject of molecular microbial ecology of animals provide a broad perspective of the variety of techniques available and their potential application in the field of animal science which is beyond the scope of this treatise. This paper will focus on nucleic acid based molecular methods which have recently been developed for studying major functional groups (cellulolytic bacteria, protozoa, fungi and methanogens) of microorganisms that are important in nutritional studies, as well as, novel methods for studying microbial diversity and function from a genomics perspective.

• 생명과학회지
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• 제18권3호
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• pp.374-380
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• 2008

본 연구는 무균성수막염 의심환자의 다양한 검체로부터 enterovirus의 진단을 위하여 real-time NASBA, 2 step RT-PCR 시험과 세포배양 시험을 각각 실시하여 각 시험법의 검출율, 특이도, 사용자 편리성, 시험소요 시간, 교차오염의 가능성 등을 비교 검토하였다. 비교시험 결과 전체 292건의 검체로부터 real-time NASBA에서 145건, 세포배양에서 101건, 2 step RT-PCR에서 86건이 양성으로 나타나 real-time NASBA가 가장 검출율이 높은 시험법임을 알 수 있었다. Enterovirus 외의 무균성수막염 원인바이러스에 대한 특이도 비교 시험결과 2 step RT-PCR 시험에서 rhinovirus 10건 중 1건이 위양성 반응을 나타내어 다른 시험법에 비해 특이도가 떨어지는 것으로 나타났다. Real-time NASBA는 하나의 튜브에서 증폭과 검출이 동시에 일어나 다른 시험과 비교하여 교차오염의 가능성이 낮으며 또한 시험 소요시간이 5시간 정도로 세포배양(5-14일 소요) 및 2 step RT-PCR(9시간소요) 에 비하여 신속하게 진단할 수 있어 일선병원이나 실험실에서 enterovirus를 검출을 위하여 적용할 수 있을 것으로 사료된다.