• Tuberculosis and Respiratory Diseases
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• 제50권5호
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• pp.599-606
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• 2001

연구배경 : 기존의 결핵진단 방법이외에 신속하고, 정확한 결핵진단법으로 결핵환자의 말초혈액에서 결핵균 PCR 검사를 시행하여 검사의 유용성과 말초혈액 결핵 PCR 검사 양성인 환자의 면역학적 상태 및 방사선학적 소견을 살펴보았다. 대상 및 방법 : 1998년 7월부터 1999년 8월까지 한림대학교 의료원에 내원하여 결핵이 의심 되었던 환자를 대상으로 하였다. 검체에서 항산균 도말검사 및 결핵균 배양 검사를 시행하였고 진단에 필요한 경우 조직검사를 시행한 환자들 중에서 3개월 이상 경과관찰이 가능하였던 59명의 환자를 대상으로 하였다. 대상 환자 모두에서 말초혈액 결핵 PCR검사를 시행하였다. 결 과 : 대상환자 59명중 남자 39예, 여자 20예였으며, 평균 연령은 44.7세였다. 45예에서 결핵으로 최종 진단되었고, 이중 41예는 결핵균 배양검사 및 조직검사로 확진되었고, 4예는 임상적으로 진단되었다. 활동성 폐결핵이 아닌 14예 중 13예는 비활동성 결핵, 1예는 폐암으로 진단되었다. 말초혈액 결핵균 PCR 양성환자는 14예였으며, 이중 활동성 결핵이 13예, 폐암이 1예였다. 이들 말초혈액 결핵균 PCR 양성인 13예의 결핵 환자의 6예(46%) 에서 면역상태의 저하를 보였다. 결핵의 진단에 있어서 말초혈액 결핵균 PCR 검사의 민감도 29%, 특이도 93%, 양성 예측도와 음성 예측도가 각각 93%, 29% 였다. 결 론 : 말초혈액 결핵균 중합효소 연쇄반응 검사는 특이도가 높은 반면 민감도는 낮아 결핵 선별 검사로는 유용하지 않을 것으로 보인다. 그러나 양성예측도가 높아 검사가 양성인 경우 활동성 결핵의 조기 진단에 도움을 줄 것으로 사료된다.

• Molecules and Cells
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• 제21권3호
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• pp.411-417
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• 2006

We have developed a polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) marker that can distinguish male-fertile (N) and male-sterile (S) cytoplasm in onions. The PCR-RFLP marker was located in a chloroplast psbA gene amplicon. Digesting the amplicons from different cytoplasm-containing varieties with the restriction enzyme MspI revealed that N-cytoplasm plants have a functional MspI site (CCGG), whereas the S-cytoplasm plants has a substitution in that site (CTGG), and thus no MspI target. The results obtained using this PCR-RFLP marker to distinguish between cytoplasmic male sterile factors in 35 onion varieties corresponded with those using a CMS-specific sequence-characterized amplified region (SCAR) marker. Moreover, the PCR-RFLP marker can identify N- ot S-cytoplasms in DNA sample mixtures in which they are in up to a 10-fold minority, indicating that use of the marker has high diagnostic precision. We also demonstrated the usefulness of the SNP detected in the psbA gene for high-throughput discrimination of CMS factors using Real-time PCR and a TaqMan probe assay.

• The Plant Pathology Journal
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• 제40권4호
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• pp.337-345
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• 2024

Soybean (Glycine max L.) is one of the most widely planted and used legumes in the world, being used for food, animal feed products, and industrial production. The soybean mosaic virus (SMV) is the most prevalent virus infecting soybean plants. This study developed a diagnostic method for the rapid and sensitive detection of SMV using a reverse transcription-recombinase polymerase amplification (RT-RPA) technique combined with a lateral flow strip (LFS). The RT-RPA and RT-RPA-LFS conditions to detect the SMV were optimized using the selected primer set that amplified part of the VPg protein gene. The optimized reaction temperature for the RT-RPA primer and RT-RPA-LFS primer used in this study was 38℃ for both, and the minimum reaction time was 10 min and 5 min, respectively. The RT-RPA-LFS was as sensitive as RT-PCR to detect SMV with 10 pg/µl of total RNA. The reliability of the developed RT-RPA-LFS assay was evaluated using leaves collected from soybean fields. The RT-RPA-LFS diagnostic method developed in this study will be useful as a diagnostic method that can quickly and precisely detect SMV in the epidemiological investigation of SMV, in the selection process of SMV-resistant varieties, on local farms with limited resources.

• Parasites, Hosts and Diseases
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• 제51권6호
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• pp.645-650
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• 2013

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at $81.5{\pm}0.2^{\circ}C$, $79.0{\pm}0.3^{\circ}C$, $76.8{\pm}0.1^{\circ}C$, and $79.9{\pm}0.1^{\circ}C$, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.

• Asian Pacific Journal of Cancer Prevention
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• 제15권15호
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• pp.6257-6261
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• 2014

Background: HPV related cervical cancer as one of the most common women cancers in developing countries. Regarding accessibility of commercial vaccines, any long or short term modality for integrating preventive immunization against HPV in a national program needs comprehensive information about HPV prevalence and its genotypes. The important role of selecting most accurate diagnostic technologies for obtaining relevant data is underlined by different assays proposed in the literature. The main objective of the present study was to introduce an in-house HPV typing assay using multiplex real time PCR with reliable results and affordable cost for molecular epidemiology surveys and diagnosis. MATERIALS AND METHODS: 112 samples of formalin fixed paraffin embedded tissues and liquid based cytology specimens from patients with known different grades of cervical dysplasia and invasive cancer, were examined by this method and the result were verified by WHO HPV LabNet proficiency program in 2013. RESULTS: HPV was detected in 105 (93.7%) out of 112 samples. The dominant types were HPV 18 (61.6%) and HPV 16 (42.9%). Among the mixed genotypes, HPV 16 and 18 in combination were seen in 12.4% of specimens. CONCLUSIONS: According to acceptable performance, easy access to primers, probes and other consumables, affordable cost per test, this method can be used as a diagnostic assay in molecular laboratories and for further planning of cervical carcinoma prevention programs.

• 한국수산과학회지
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• 제54권5호
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• pp.691-697
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• 2021

Vibrio parahaemolyticus associated with acute hepatopancreatic necrosis disease (Vp_AHPND) and Enterocytozoon hepatopenaei (EHP) are the two most important pathogens in shrimp aquaculture and they have caused enormous losses to the shrimp industry worldwide. In ponds, the major target organ for the two pathogens is the hepatopancreas, and infection with EHP is a known potential risk factor for Vp_AHPND infection. This study aimed to develop a PCR (polymerase chain reaction)-based diagnostic method for simultaneously detecting Vp_AHPND and EHP. The newly developed PCR diagnostic method could be used to test various samples, such as seawater, shrimp, and feces. The diagnostic method exhibited high sensitivity and specificity for both pathogens. This will help reduce the potential economic losses that may have been caused by the two major shrimp pathogens, Vp_AHPND and EHP, and will allow for the efforts and time spent combatting them to be dedicated elsewhere.

• 한국동물위생학회지
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• 제41권1호
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• pp.29-39
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• 2018

In this study, we developed a sensitive and specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid visual detection of foot-and-mouth disease virus (FMDV) circulated in Korea. The RT-LAMP was completed in 40 min at $62^{\circ}C$ and the results of the assay were directly detected by naked eye without any detection process. The assay specifically amplified all 7 serotypes of FMDV RNAs but not amplified other viral and cellular nucleic acids. The sensitivity of the RT-LAMP was $10^2$, $10^3$ and $10^3TCID_{50}/mL$ for serotype O, A and Asia 1 FMDV, respectively, which was comparable to conventional reverse transcription polymerase chain reaction (RT-PCR) and relatively lower than that of real time quantitative RT-PCR (qRT-PCR). Clinical evaluation of the RT-LAMP using different serotypes of Korean and foreign FMDV strains showed a 100% (35/35) agreement with the results of the RT-PCR and qRT-PCR. These results indicated that RT-LAMP assay developed in this study could be a valuable diagnostic method for FMDV monitoring and surveillance.

• Parasites, Hosts and Diseases
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• 제54권3호
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• pp.329-334
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• 2016

Trichomoniasis caused by Trichomonas vaginalis is a common sexually transmitted disease. Its association with several health problems, including preterm birth, pelvic inflammatory disease, cervical cancer, and transmission of human immunodeficiency virus, emphasizes the importance of improved access to early and accurate detection of T. vaginalis. In this study, a rapid and efficient loop-mediated isothermal amplification-based method for the detection of T. vaginalis was developed and validated, using vaginal swab specimens from subjects suspected to have trichomoniasis. The LAMP assay targeting the actin gene was highly sensitive with detection limits of 1 trichomonad and 1 pg of T. vaginalis DNA per reaction, and specifically amplified the target gene only from T. vaginalis. Validation of this assay showed that it had the highest sensitivity and better agreement with PCR (used as the gold standard) compared to microscopy and multiplex PCR. This study showed that the LAMP assay, targeting the actin gene, could be used to diagnose early infections of T. vaginalis. Thus, we have provided an alternative molecular diagnostic tool and a point-of-care test that may help to prevent trichomoniasis transmission and associated complications.

• 한국동물위생학회지
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• 제30권4호
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• pp.489-496
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• 2007

An oligonucleotide microarray system was developed for the simultaneous detection of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine enteric calicivirus, porcine group A and C rotavirus. RNAs of the reference viruses and porcine diarrhea samples were extracted and amplified using one-step multiplex RT-PCR in the presence of cyanine 5-dCTP and hybridized on the microarray chip that spotted the virus-specific oligonucleotides. This system were approximately 10-to 100-fold higher in sensitivity than conventional RT-PCR, and the assay time was less than 3 hours. The relative sensitivity and specificity were 92% and 72.2%, respectively, based on 102 porcine diarrhea samples using RT-PCR as gold standard. These results suggested that the oligonucleotide microarray system in this study be probably more reliable and reproducible means for detecting porcine enteric viruses and that it could be of substantial use in routine diagnostic laboratories.

• Preventive Nutrition and Food Science
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• 제9권3호
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• pp.276-282
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• 2004

Marginal Zn deficiency is prevalent through the world and yet human zinc status has not been properly assessed due to the lack of a reliable diagnostic indicator. One potential possibility for zinc status assessment using Zn-binding protein, metallothionein (MT)-mRNA, has been proposed. The purpose of the present study was aimed to show whether measurement of mononuclear cell (MNC) MT mRNA, using a competitive-reverse transcriptase-polymerase chain reaction (competitive-RT-PCR) assay, could indicate zinc status in human subjects. In this study, MNC MT-mRNA expression was measured using a competitive-RT-PCR to compare before and after 14 days of zinc supplementation (50 mg Zn/das zinc gluconate). RT-PCR oligonucleotide primers which were designed to amplify both a 278 bp segment of the human MT-2A cDNA and a 198 bp mutant competitor cDNA template from MNCs, were prepared. MT-2A mRNA was normalized by reference to the housekeeping gene, $\beta$-actin, mRNA for which was also measured by competitive-RT-PCR. There was considerable inter-individual variation in MT-mRNA concentration and yet, the mean MT-2A mRNA level increased 4.7-fold after Zn supplementation, as compared to before Zn supplementation. This MT-2A mRNA level was shown as the same pattern and, even more sensitive assay, compared to the conventional plasma and red blood cells (RBCs) Zn assessment in which plasma and RBCs zinc levels increased 2.3- and 1.2-fold, respectively (p<0.05). We suggest that MT competitive-RT-PCR can be a useful assessment tool for evaluating human zinc status.