• The Plant Pathology Journal
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• 제30권4호
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• pp.416-424
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• 2014

Sweet potato is grown extensively from tropical to temperate regions and is an important food crop worldwide. In this study, we established detection methods for 17 major sweet potato viruses using single and multiplex RT-PCR assays. To investigate the current incidence of viral diseases, we collected 154 samples of various sweet potato cultivars showing virus-like symptoms from 40 fields in 10 Korean regions, and analyzed them by RT-PCR using specific primers for each of the 17 viruses. Of the 17 possible viruses, we detected eight in our samples. Sweet potato feathery mottle virus (SPFMV) and sweet potato virus C (SPVC) were most commonly detected, infecting approximately 87% and 85% of samples, respectively. Furthermore, Sweet potato symptomless virus 1 (SPSMV-1), Sweet potato virus G (SPVG), Sweet potato leaf curl virus (SPLCV), Sweet potato virus 2 ( SPV2), Sweet potato chlorotic fleck virus (SPCFV), and Sweet potato latent virus (SPLV) were detected in 67%, 58%, 47%, 41%, 31%, and 20% of samples, respectively. This study presents the first documented occurrence of four viruses (SPVC, SPV2, SPCFV, and SPSMV-1) in Korea. Based on the results of our survey, we developed multiplex RT-PCR assays for simple and simultaneous detection of the eight sweet potato viruses we recorded.

• 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.

• BMB Reports
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• 제38권5호
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• pp.555-562
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• 2005

Single nucleotide polymorphisms (SNPs) are becoming the most common type of markers used in genetic analysis. In the present report a SNP has been chosen to test the applicability of Real Time PCR to discriminate and quantify SNPs alleles on DNA pools. Amplification Refractory Mutation System (ARMS) and Mismatch Amplification Mutation Assay (MAMA) has been applied. Each assay has been pre-validated testing specificity and performances (linearity, PCR efficiency, interference limit, limit of detection, limit of quantification, precision and accuracy). Both the approaches achieve a precise and accurate estimation of the allele frequencies on pooled DNA samples in the range from 5% to 95% and don't require standard curves or calibrators. The lowest measurement that could be significantly distinguished from the background noise has been determined around the 1% for both the approaches, allowing to extend the range of quantifications from 1% to 99%. Furthermore applicability of Real Time PCR assays for general diagnostic purposes is discussed.

• Parasites, Hosts and Diseases
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• 제51권5호
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• pp.573-577
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• 2013

A loop-mediated isothermal amplification (LAMP) assay allows rapid diagnosis of Toxoplasma gondii infection. In the present study, the LAMP assay was evaluated using blood from both naturally and experimentally infected pigs. The sensitivity of the LAMP assay was compared with that of Q-PCR. Both assays detected T. gondii in the blood of experimentally infected pigs, with 100% agreement. In infected blood samples, the parasite was detected as early as 2 days post-infection and reached a peak in 3-5 days. In 216 field serum samples, the detection rates of LAMP and Q-PCR assays were 6.9% and 7.8%, respectively. This result indicates that the sensitivity of the LAMP assay was slightly lower than that of the Q-PCR assay. However, the LAMP may be an attractive diagnostic method in conditions where sophisticated and expensive equipment is unavailable. This assay could be a powerful supplement to current diagnostic methods.

• 한국동물위생학회지
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• 제46권2호
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• pp.123-135
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• 2023

Feline calicivirus (FCV) is considered the main viral pathogen of feline upper respiratory tract disease (URTD). The frequent mutations of field FCV strains result in the poor diagnostic sensitivity of previously developed molecular diagnostic assays. In this study, a more sensitive real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for broad detection of currently circulating FCVs and comparatively evaluated the diagnostic performance with previously developed qRT-PCR assay using clinical samples collected from Korean cat populations. The developed qRT-PCR assay specifically amplified the FCV p30 gene with a detection limit of below 10 copies/reaction. The assay showed high repeatability and reproducibility, with coefficients of intra-assay and inter-assay variation of less than 2%. Based on the clinical evaluation using 94 clinical samples obtained from URTD-suspected cats, the detection rate of FCV by the developed qRT-PCR assay was 47.9%, which was higher than that of the previous qRT-PCR assay (43.6%). The prevalence of FCV determined by the new qRT-PCR assay in this study was much higher than those of previous Korean studies determined by conventional RT-PCR assays. Due to the high sensitivity, specificity, and accuracy, the new qRT-PCR assay developed in this study will serve as a promising tool for etiological and epidemiological studies of FCV circulating in Korea. Furthermore, the prevalence data obtained in this study will contribute to expanding knowledge about the epidemiology of FCV in Korea.

• Tuberculosis and Respiratory Diseases
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• 제62권5호
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• pp.406-416
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• 2007

연구배경: 단일염기다형성(Single nucleotide polymorphism, SNP)은 인간의 유전자 서열 1000염기에 1개 빈도로 발견되어 인간은 대략 300만개의 유전자 다형성을 가지고 있다. 이 유전자 다형성의 조합결과로 인간의 개체 간 특성들이 결정되는 것으로 이해되고 있다. 이러한 다형성들의 조합양상에 따라 특이 질환에 대한 유전자 감수성 또한 달라지게 되므로 최근에는 많은 질환들과 유전자 다형성들과의 상관관계를 보는 연구들도 활발하게 진행되고 있다. 이러한 SNP분석은 큰 집단을 대상으로 진행되어 지므로 적은 비용으로 정확하게 그리고 대용량으로 분석할 수 있는 방법이 필요하다. 방 법: 대상 환자 89명의 genomic DNA를 가지고서 promotor상에 위치한 -37과 -524 염기부위에서 유전자 다형성을 보이는 것으로 보고되어져 있는 RRM1(ribonucleotide reductase M1) 유전자를 대상으로 PCR-RFLP(polymerase chain reaction-restriction fragment length polymorphism)와 real-time PCR(RTPCR, TaqMan probe assay)을 동시에 시행한 후 각각의 결과를 비교 분석하였다. 결 과: 대상 DNA 89예 중 -37에서는 2예(2.17%), -524에서는 15예(16.26%)가 서로 다른 양상을 보였다. 결과 차이를 보인 샘플 17예를 대상으로 직접 염기서열 분석을 시행하여 본 결과, 17예 모두 RT-PCR에서 확인되었던 결과와 일치함을 확인할 수 있었다. 추가 샘플 138예를 대상으로 RT-PCR을 2회 연속 실행하여 genotyping을 해 본 결과 98%이상의 높은 일치율을 보였으며, 그중 10예를 무작위로 골라 직접 염기서열 분석을 시행하여 본 결과, 역시 100%일치, 높은 정확도를 보였고 이는 in-tube assay 방식으로 샘플의 오염을 최소화 할 수 있었으며 72 well based system(Corbett Research)을 이용함으로 1회 유전자 증폭반응을 통해 많은 검체를 한 번에 확인할 수 있어 매우 빠른 검사방법 이었다. 결 론: 큰 집단을 대상으로 다량의 SNP를 분석하기 위한 실험 방법으로는 RT-PCR이 신속하면서도 정확한 결과를 얻을 수 있는 방법으로 사료된다.

• 대한임상검사과학회지
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• 제48권2호
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• pp.88-93
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• 2016

선형 사상충의 일종인 심장사상충은 개의 심폐 사상충증을 유발한다. 이에 본 연구의 목적은 심장사상충을 효과적으로 검출할 수 있는 실시간 중합효소연쇄반응 기법을 개발함에 있다. 연구에 있어서 사용된 프라이머 및 프로브는 선행연구에서 제작된 심장사상충 특이 프라이머 및 새롭게 제작된 TaqMan 프로브를 이용하였다. 선행연구에서 제작된 프라이머 및 농도별로 희석된 게놈유전자와 플라스미드유전자가 SYBR Green 실시간 중합효소연쇄반응 수행에 이용되었으며, 중합효소연쇄반응 과정 중 증폭 이후의 녹는 곡선의 결과를 분석하였다. 분석결과 사용된 프라이머는 각각 게놈유전자 및 플라스미드 유전자에서 특이 녹는 곡선을 나타냄에 따라 심장사상충 특이 사이토크롬 C 산화효소 유전자만을 증폭하고 있음을 확인 할 수 있었다. 새롭게 제작된 TaqMan 프로브는 SYBR Green 실시간 중합효소연쇄반응과의 결과를 농도별로 희석된 플라스미드 유전자를 이용하여 비교 분석하였고, 분석결과 TaqMan 프로브를 이용한 실시간 중합효소연쇄반응이 검출효율 및 특이도에 있어서 우수함을 확인할 수 있었다. 본 연구를 통하여 개발한 실시간 중합효소연쇄반응은 기존의 전통적인 진단기법의 한계를 극복할 수 있는 신속하고 정확한 향상된 진단기법을 제시한다.

• Journal of Microbiology and Biotechnology
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• 제33권8호
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• pp.1091-1100
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• 2023

Human papillomavirus (HPV) types 16 and 18 are the major causes of cervical lesions and are associated with 71% of cervical cancer cases globally. However, public health infrastructures to support cervical cancer screening may be unavailable to women in low-resource areas. Therefore, sensitive, convenient, and cost-efficient diagnostic methods are required for the detection of HPV16/18. Here, we designed two novel methods, real-time ERA and ERA-LFD, based on enzymatic recombinase amplification (ERA) for quick point-of-care identification of the HPV E6/E7 genes. The entire detection process could be completed within 25 min at a constant low temperature (35-43℃), and the results of the combined methods could be present as the amplification curves or the bands presented on dipsticks and directly interpreted with the naked eye. The ERA assays evaluated using standard plasmids carrying the E6/E7 genes and clinical samples exhibited excellent specificity, as no cross-reaction with other common HPV types was observed. The detection limits of our ERA assays were 10⁰ and 10¹ copies/µl for HPV16 and 18 respectively, which were comparable to those of the real-time PCR assay. Assessment of the clinical performance of the ERA assays using 114 cervical tissue samples demonstrated that they are highly consistent with real-time PCR, the gold standard for HPV detection. This study demonstrated that ERA-based assays possess excellent sensitivity, specificity, and repeatability for HPV16 and HPV18 detection with great potential to become robust diagnostic tools in local hospitals and field studies.

• Parasites, Hosts and Diseases
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• 제57권1호
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• pp.27-31
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• 2019

PCR is known to be the most sensitive method for diagnosing Trichomonas vaginalis infections. This study aimed to compare the sensitivity of a PCR assay for trichomoniasis (HY-PCR) developed in Hanyang University with the use of a Seeplex Ace Detection $Kit^{(R)}$, using urine collected from four Korean men with prostatic disease. Overall, HY-PCR was more sensitive than the Seeplex Kit. The use of Chelex 100 is recommended for DNA isolation in order to increase the sensitivity of the PCR test.

• 한국동물위생학회지
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• 제39권2호
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• pp.107-116
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• 2016

In the study, we developed and evaluated a uracil N-glycosylase (UNG)-supplemented single-tube nested reverse transcription-polymerase chain reaction (UsnRT-PCR) assay that can carried out first-round RT-PCR and second-round nested PCR in a reaction tube without reaction tube opening and can simultaneously detect EU- and NA-PRRSV. The UsnRT-PCR confirmed to have a preventing ability of mis-amplification by contamination of pre-amplified PRRSV DNA from previous UsnRT-PCR. Primer specificities were evaluated with RNAs extracted from 8 viral strains and our results revealed that the primers had a high specificity for both genotypes of PRRSV. The sensitivity of the UsnRT-PCR was 0.1 $TCID_{50}$/0.1 mL for EU- or NA-PRRSV, respectively, which is comparable to that of previously reported real time RT-PCR (RRT-PCR). Clinical evaluation on 110 field samples (60 sera and 50 lung tissues) by the UsnRT-PCR and the RRT-PCR showed that detection rates of the UsnRT-PCR was 70% (77/110), and was relatively higher than that of the RRT-PCR (69.1%, 76/110). The percent positive or negative agreement of the UsnRT-PCR compared to RRT-PCR was 96.1% (73/76) or 90.9% (30/33), showing that the test results of both assays may be different for some clinical samples. Therefore, it is recommend that diagnostic laboratory workers use the two diagnostic assays for the correct diagnosis for the relevant samples in the swine disease diagnostic laboratories. In conclusion, the UsnRT-PCR assay can be applied for the rapid, and reliable diagnosis of PRRSV without concerns about preamplified DNA carryover contamination that can occurred in PCR process in the swine disease diagnostic laboratories.