• Journal of Microbiology and Biotechnology
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• 제19권11호
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• pp.1464-1469
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• 2009

To achieve more accurate and rapid detection of macrolide-lincosamide-streptogramin B resistance genes, erm(A), erm(B), and erm(C), we developed a TaqMan probe-based real-time PCR (Q-PCR) method and compared it with conventional PCR (C-PCR), which is the most widely using erm gene identification method. The detection limit of Q-PCR was 5 fg of genomic DNA or 5-8 CFU of bacterial cells of Staphylococcus aureus. The utilization of Q-PCR might shorten the time to erm detection from 3-4 h to about 50 min. These data indicated that Q-PCR assay appears to be not only highly sensitive and specific, but also the most rapid diagnostic method. Therefore, the appropriate application of the Q-PCR assay will permit rapid and accurate identification of erm genes from clinical and other samples.

• 대한의생명과학회지
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• 제24권3호
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• pp.230-238
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• 2018

Human enteric Adenovirus-41 (HuEAdV-41) causes gastroenteritis, which detected by the polymerase chain reaction (PCR) base diagnostic system for clinical, food, environmental, fish and shellfish samples. We developed improved PCR and nested PCR primer set which had high specificity, sensitivity and reduced times. In this study, we compared seventeen conditions reported in the previous study that was using the PCR based HuEAdV-41 detection system, and non-enteric Adenovirus were detected in nine conditions. The most sensitive detection condition was up to 25 copies however it took 184 minutes of PCR reaction time. In this study, the PCR primer set developed had same level of sensitivity, it reduced the time of detection for clinical, food and seafood samples to 112 minutes. Developed nested PCR primer set needed 112 minutes but detected up to approximately 1 copy. In addition, developed PCR and nested PCR primer set was validated with twenty samples of underground water at random, of which ten samples showed specific band without non-specific reaction. We expect this study will be used to diagnose HuEAdV-41 from various samples.

• 대한수의학회지
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• 제42권4호
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• pp.495-504
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• 2002

Streptococcus suis is an important swine pathogen in nearly all countries with an extensive pig industry. It is associated with meningitis, arthritis, endocarditis, septicaemia, bronchopneumonia and sudden death. Attempts to control the disease are still hampered the lack of effective vaccines and sensitive diagnostic tools. A PCR method which can be used for the detection of virulent strains of serotype 2, which is most prevalent serotype, and serotype 1 was developed. However, serotype 1, 2, 7 and 9 strains are frequently isolated from diseased pigs. In European countries, S suis serotype 2 is the most prevalent type isolated from diseased pigs, followed by serotype 9 and 1. In Japan, capsular serotype 2 was also the most prevalent serotype, followed by capsular serotype 7. Most of S suis isolated from diseased pigs belong to a limited number of capsular serotype, often those between 1 and 9. We investigated the distribution of S suis serotype 1, 2, 7 and 9 from 740 pig lungs at abattoir in Jeolla and Chungcheong by rapid multiplex PCR assay. Fifty of 740 lung samples, 6.8%, were S suis postitive and identified S suis were divided by 38% (19/50) in serotype 2, 2% (1/50) in serotype 7 and 4% (2/50) in serotype 9. The distribution of S suis serotype in Korea was similar to other countries. Moreover, the multiplex PCR assay may be an useful diagnostic tool for the detection of pigs carrying serotype 1, 2, 7, 1/2, 9 and 14 strains in epidemiological and transmission studies and facilitate control and eradication programs.

• Parasites, Hosts and Diseases
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• 제54권4호
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• pp.393-397
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• 2016

Low parasitemic condition in malaria remains a diagnostic challenge; as the available diagnostic methods failed to detect. Currently, hemozoin (Hz) pigment is gaining attention in the diagnosis of malaria. The major drawback is ease of detection of Hz in routine practice. A pilot study was conducted to evaluate the role of Hz pigment and to compare the performance of quantitative buffy coat assay (QBC) and PCR in such conditions. Clinically suspected cases of malaria were examined by both Giemsa stain and immunochromatographic test (ICT). Samples positive by ICT and negative by Giemsa stain were further examined by nested PCR targeting 18S rRNA and QBC for the presence of malaria parasites and pigments. Thirty blood samples fulfilled the inclusion criteria out of which 23 were Plasmodium vivax (Pv), 4 Plasmodium falciparum (Pf), and 3 mixed (Pv and Pf) by immunochromatographic test. Twenty-one out of 30 (70%) were positive by nested PCR in comparison to 25/30 (83%) by QBC. Samples containing both malaria parasites and Hz pigment by QBC completely showed concordance with the PCR result. However, 61% of total samples containing only Hz pigment were observed positive by PCR. Hz pigment remains an important tool for malaria diagnosis. Identification of leukocytes containing pigments by QBC not only indicates recent malarial infections but also puts light on severity of the disease. QBC assay is a rapid, highly sensitive, and cost-effective method to detect malaria parasites and Hz pigment especially in low parasitemic conditions.

• 한국양봉학회지
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• 제34권1호
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• pp.39-46
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• 2019

BQCV multi-point PCR was developed as a rapid multiplex detection method for BQCV, one of the viral pathogens of honeybees. It could detect BQCV specific genes qualitative as well as quantitative detection based on ultra-rapid PCR. Three primer pairs (RNA dependent RNA polymerase, capsid protein, 3C like protease) were specifically designed for accurate the detection and were optimized for minimizing the detection time and increasing the sensitivity. Our advanced diagnostic system have the accuracy by lowering the concern about the variation in the BQCV detection site. In addition, it should be an opportunity to identify mutations that are mixed with other viruses.

• 대한의생명과학회지
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• 제26권3호
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• pp.170-178
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• 2020

Mycobacterium tuberculosis (MTB) continues to be one of the main causative agents of tuberculosis (TB); moreover, the incidence of nontuberculous mycobacteria (NTM) infections has been rising gradually in both immunocompromised and immunocompetent patients. Precise and rapid detection and identification of MTB and NTM in respiratory specimens are thus important for MTB infection control. Molecular diagnostic methods based on the nucleic acid amplification test (NAAT) are known to be rapid, sensitive, and specific compared to the conventional acid-fast bacilli (AFB) smear and mycobacterial culture methods. In the present study, the clinical performances of three commercial molecular diagnostic assays, namely TB/NTM PCR (Biocore), MolecuTech Real MTB-ID^® (YD Diagnostics), and REBA Myco-ID^® (YD Diagnostics), were evaluated with a total of 92 respiratory specimens (22 AFB smear positives and 67 AFB smear negatives). The sensitivity and specificity of TB/NTM PCR were 100% and 75.81%, respectively. The corresponding values of MolecuTech Real MTB-ID^® and REBA Myco-ID^® were 56.52% and 90.32%, and 56.52% and 82.26%, respectively. TB/NTM PCR showed the highest sensitivity; however, the concordant rate was 10% compared with sequence analysis. Although MolecuTech Real MTB-ID^® showed lower sensitivity, its specificity was the highest among the three methods. REBA Myco-ID^® allowed accurate classification of NTM species; therefore, it was the most specific diagnostic method. Of the three PCR-based methods, MolecuTech Real MTB-ID^® showed the best performance. This method is expected to enable rapid and accurate identification of MTB and NTM.

• Parasites, Hosts and Diseases
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• 제48권2호
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• pp.99-103
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• 2010

Malaria is endemic in the Cukurova region while it is sporadic in other regions of Turkey. Therefore, the laboratory and clinical diagnosis of malaria is important for the treatment of malaria. In this study, 92 blood samples that were taken from the suspected malaria patients for routine diagnosis in a period of 10 years between 1999 and 2009 were analyzed. All of these blood samples were examined by microscopic examinations using Giemsa-stained thick blood films, nested PCR, and real-time PCR. The sensitivity-specificity and positive-negative predictive values for these diagnostic tests were then calculated. It was found that the positive predictive values of microscopic examination of thick blood films, nested PCR, and real-time PCR were 47.8%, 56.5%, and 60.9% for malaria, respectively. The real-time PCR was found to have a specificity of 75% and sensitivity of 100%, while specificity and sensitivity of nested PCR was found 81.2% and 97.7% according to the microscopic examination of thick blood films, respectively.

• 한국수의병리학회지
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• 제6권1호
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• pp.1-5
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• 2002

A1celaphine herpesvirus 1 (AHV-1) is a causative agent of malignant catarrhal fever which is a fatal and a lymphoproliferative syndrome. AHV-1 is a gamma herpesvirus, which induces frequent latent infection and often difficult to detect its antigens or specific nucleic acids because of its low viral copies in the infected tissues. A new method, in situ PCR, is developed for the detection of AHV-1 nucleic acid in this study. Target sequences of AHV-1 open reading frame 50 gene were detected within AHV-1 infected MDBK cells. As compare with other molecular biological methods for the detection of AHV-1, in situ PCR was found to be more sensitive than in situ hybridization and to be less sensitive than nested PCR. However, nested PCR cannot afford to observe and differentiate AHV-1 infected cells. In situ PCR amplifies a target sequence within cells that can be visualized microscopically with increased sensitivity compared to detection by in situ hybridization. In situ PCR has wide applications for sensitive localization of low copy AHV-1 viral sequences within cells to investigate the role of viruses in a variety of clinical conditions and also provide the rapid, sensitive, and specific detection of AHV-1 infection.

• 대한의생명과학회지
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• 제25권4호
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• pp.426-430
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• 2019

Currently, molecular diagnostic assays based on nucleic acid amplification tests have been shown to effectively detect mycobacterial infections in various types of specimen, however, variable sensitivity was shown in FFPE samples according to the kind of commercial kit used. The present study therefore used automated PCR-reverse blot hybridization assay (REBA) system, REBA Myco-ID HybREAD 480^®, for the rapid identification of Mycobacterium species in various types of human tissue and compared the conventional one-tube nested-PCR assay for detecting Mycobacterium tuberculosis (MTB). In conventional nested-PCR tests, 25 samples (48%) were MTB positive and 27 samples (52%) were negative. In contrast, when conducted PCR-REBA assay, 11 samples (21%) were MTB positive, 20 samples (39%) were NTM positive, 8 samples (15%) were MTB-NTM double positive, and 13 samples (25%) were negative. To determine the accuracy and reliability of the two molecular diagnostic tests, the one-tube nested-PCR and PCR-REBA assays, were compared with histopathological diagnosis in discordant samples. When conducted nested-PCR assay, 10 samples (59%) were MTB positive and seven samples (41%) were negative. In contrast, when conducted PCR-REBA test, three samples (17%) were MTB positive, 10 samples (59%) were NTM positive and four samples (24%) were negative. In conclusion, the automated PCR-REBA system proved useful to identify Mycobacterium species more rapidly and with higher sensitivity and specificity than the conventional molecular assay, one-tube nested-PCR; it might therefore be the most suitable tool for identifying Mycobacterium species in various types of human tissue for precise and accurate diagnosis of mycobacterial infection.

• 한국식물병리학회지
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• 제13권4호
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• pp.219-224
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• 1997

여러 계통의 PVY 외피단백질 유전자간에 상동성이 높은 부위에 위치한 primer 쌍을 이용하여 RT-PCR 방법으로 PVY 검정법을 개발하였는데 여러 line의 대서품종으로부터 764 bp의 PCR 산물이 합성되었고 이 절편을 sequencing한 결가 PVY의 유전자임을 확인하였다. 싹과 괴경 조직에서 RT-PCR에 의한 PVY의 검정의 민감도는 ELISA 방법보다 약 1,000배정도 높았다.