• Parasites, Hosts and Diseases
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• 제60권4호
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• pp.295-299
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• 2022

Malaria elimination and control require prompt and accurate diagnosis for treatment plan. Since microscopy and rapid diagnostic test (RDT) are not sensitive particularly for diagnosing low parasitemia, highly sensitive diagnostic tools are required for accurate treatment. Molecular diagnosis of malaria is commonly carried out by nested polymerase chain reaction (PCR) targeting 18S rRNA gene, while this technique involves long turnaround time and multiple steps leading to false positive results. To overcome these drawbacks, we compared highly sensitive cytochrome oxidase gene-based single-step multiplex reaction with 18S rRNA nested PCR. Cytochrome oxidase (cox) genes of P. falciparum (cox-III) and P. vivax (cox-I) were compared with 18S rRNA gene nested PCR and microscopy. Cox gene multiplex PCR was found to be highly specific and sensitive, enhancing the detection limit of mixed infections. Cox gene multiplex PCR showed a sensitivity of 100% and a specificity of 97%. This approach can be used as an alternative diagnostic method as it offers higher diagnostic performance and is amenable to high throughput scaling up for a larger sample size at low cost.

• 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.

• Parasites, Hosts and Diseases
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• 제53권2호
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• pp.147-154
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• 2015

Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (${\approx}375bp$) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects ${\approx}550bp$ of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, ${\approx}2$ oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.

• Journal of Microbiology and Biotechnology
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• 제24권7호
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• pp.1004-1007
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• 2014

Culture is the gold standard for diagnosis of tuberculosis, but it takes 6 to 8 weeks to confirm the result. This issue is complemented by the detection method using polymerase chain reaction, which is now widely used in a routine microbiology laboratory. In this study, we evaluated the performance of the Seegene Anyplex TB PCR to assess its diagnostic sensitivity and specificity, and compared its results with the Roche Cobas TaqMan MTB PCR, one of the most widely used assays in the world. Five university hospitals located in the Chungcheong area in South Korea participated in the study. A total of 1,167 respiratory specimens ordered for acid-fast bacilli staining and culture were collected for four months, analyzed via the Seegene Anyplex TB PCR, and its results were compared with the Roche Cobas TaqMan MTB PCR. For detection of Mycobacterium tuberculosis, the diagnostic sensitivity and specificity of the Anyplex TB PCR were 87.5% and 98.2% respectively, whereas those of the Cobas TaqMan were 92.0% and 98.0% respectively (p value > 0.05). For smear-positive specimens, the sensitivity of the Anyplex TB PCR was 95.2%, which was exactly the same as that of the Cobas TaqMan. For smear-negative specimens, the sensitivity of the Anyplex TB PCR was 69.2%, whereas that of the Cobas TaqMan TB PCR was 84.6%. For detection of MTB, the Seegene Anyplex TB PCR showed excellent diagnostic performance, and high sensitivity and specificity, which were comparable to the Roche Cobas TaqMan MTB PCR. In conclusion, the Anyplex TB PCR can be a useful diagnostic tool for the early detection of tuberculosis in clinical laboratories.

• 대한수의학회지
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• 제43권3호
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• pp.471-476
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• 2003

Malignant catarrhal fever (MCF) is a systemic disease of ruminants caused by ovine herpesvirus 2 (OvHV-2). OvHV-2 is a gamma herpesvirus, which induces frequent latent infection and often difficult to detect its antigens and even specific nucleic acids because of its low viral copies in the infected tissues. Histopathology, serology and polymerase chain reaction (PCR) were compared for the diagnosis of MCF using 10 bison infected with OvHV-2. Histopathological diagnosis was performed using the criteria which was based upon the pathognomic lesions. Serological diagnosis was conducted using its serum with competitive ELISA for the detection of antibodies of OvHV-2. Also, the nest PCR was performed with peripheral blood leukocytes for the detection of OvHV-2-specific DNAs. Primers 556 and 775 were used for the primary amplification, and primers 556 and 555 were used for the secondary amplification. As the results, positive cases were 6 by histopahology, 9 by serology and 10 by PCR. As comparing with other diagnostic methods, PCR was found to be more sensitive than histopathology and serology. The recent development of molecular diagnostic assays has provided powerful tools for investigating how viruses survive in nature. Development of PCR specific for viruses has dramatically improved the accuracy of diagnosis of viruses in clinically infected animals. Furthermore, amplification of viral genomic material by nest PCR represents the most sensitive method for the detection of viruses and might be detected successfully even though very low viral DNA copies. So, it could be used as the first choice for the detection of viral DNAs with low copies such as the status of latent infection. However, it has also some limitation of application like as false negative results by PCR inhibitors and false positive results by contamination. The results of this study suggest that the use of molecular biological methods like PCR may increase the accuracy for the diagnosis of infectious diseases. However, in diagnostic laboratory, it is recommended that PCR assay must be conducted with other diagnostic methods for more reliable diagnosis.

• Fisheries and Aquatic Sciences
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• 제24권11호
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• pp.351-359
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• 2021

The red sea bream iridovirus (RSIV) belonging to genus Megalocytivirus is responsible for red sea bream iridoviral disease (RSIVD) in marine and freshwater fishes. Although several diagnostic assays for RSIV have been developed, diagnostic sensitivity (DSe) and specificity (DSp) of real-time polymerase chain reaction (PCR) assays are not yet evaluated. In this study, we developed a TaqMan probe-based real-time PCR method and evaluated its DSe and DSp. To detect RSIV, the probe and primers were designed based on consensus sequences of the major capsid protein (MCP) genes from megalocytiviruses including RSIV, infectious spleen and kidney necrosis virus (ISKNV), and turbot reddish body iridovirus (TRBIV). The probe and primers were shown to be specific for RSIV, ISKNV, and TRBIV-types megalocytiviruses. A 95% limit of detection (LOD_95%) was determined to be 5.3 viral genome copies/µL of plasmid DNA containing the MCP gene from RSIV. The DSe and DSp of the developed real-time PCR assay for field samples (n = 112) were compared with those of conventional PCR assays and found to be 100% and 95.2%, respectively. The quantitative results for SYBR Green and TaqMan probe-based real-time PCR were not significantly different. The TaqMan probe-based real-time PCR assay for RSIV may be used as an appropriate diagnostic tool for qualitative and quantitative analysis.

• 한국임상수의학회지
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• 제20권1호
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• pp.7-11
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• 2003

감염된 환축을 찾는 진단과정에 완벽하지 못한 진단검사를 사용하는 경우 진단검사 결과는 흔히 왜곡되어 나타난다. 본 연구에서 저자는 Helicobacter pylori 감염을 진단하는데 사용되는 urease 검사, PCR 검사 및 조직학적인 검사법을 단독으로 사용하는 경우와 병행하여 사용하는 상황으로 구분하여 각각 진단적 특성을 평가하였다. 비선형 회귀모형 분석결과 민감도, 특이도, 양성우도비 및 음성우도비는 urease 검사법의 경우 99.9%, 99.9%, 99.9%, 99.6%, PCR 검사의 경우 88.6%, 99.9%, 99.9%, 70.5%, 조직검사법의 경우 78.3%, 97%, 78.3%, 97%fh 나타났다. 예측도는 유병율의 변화에 따라 다양한 값을 보였으며 Helicobacter pylori 감염의 유병율이 35% 이상일 때 조직 검사상 양성결과는 90% 이상의 일치도를 보였고, 유병율이 25% 미만일 때 조직 검사상 음성결과는 90% 이상의 일치도를 보였다. 본 연구결과 임상에서 감염된 개체를 스크리닝하는 목적으로 세가지 진단검사를 병행하는 것은 실질적인 이익이 없으며 단독검사로서 urease 검사와 PCR 검사가 가장 적합한 것으로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제33권11호
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• pp.1457-1466
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• 2023

Enteric fever is caused by typhoidal Salmonella serovars (Typhi, Paratyphi A, Paratyphi B, and Paratyphi C). Owing to the importance of Salmonella serovars in clinics and public hygiene, reliable diagnostics for typhoidal serovars are crucial. This study aimed to develop a novel diagnostic tool for typhoidal Salmonella serovars and evaluate the use of human blood for clinically diagnosing enteric fever. Five genes were selected to produce specific PCR results against typhoidal Salmonella serovars based on the genes of Salmonella Typhi. Heptaplex PCR, including genetic markers of generic Salmonella, Salmonella enterica subsp. enterica, and typhoidal Salmonella serovars, was developed. Typhoidal Salmonella heptaplex PCR using genomic DNAs from 200 Salmonella strains (112 serovars) provided specifically amplified PCR products for each typhoidal Salmonella serovar. These results suggest that heptaplex PCR can sufficiently discriminate between typhoidal and non-typhoidal Salmonella serovars. Heptaplex PCR was applied to Salmonella-spiked blood cultures directly and provided diagnostic results after 12- or 13.5-h blood culture. Additionally, it demonstrated diagnostic performance with colonies recovered from a 6-h blood culture. This study provides a reliable DNA-based tool for diagnosing typhoidal Salmonella serovars that may be useful in clinical microbiology and epidemiology.

• Parasites, Hosts and Diseases
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• 제59권1호
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• pp.77-82
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• 2021

As malaria remains a major health problem worldwide, various diagnostic tests have been developed, including microscopy-based and rapid diagnostic tests. LabChip real-time PCR (LRP) is a small and portable device used to diagnose malaria using lab-on-a-chip technology. This study aimed to evaluate the diagnostic performance of LRP for detecting malaria parasites. Two hundred thirteen patients and 150 healthy individuals were enrolled from May 2009 to October 2015. A diagnostic detectability of LRP for malaria parasites was compared to that of conventional RT-PCR. Sensitivity of LRP for Plasmodium vivax, P. falciparum, P. malariae, and P. ovale was 95.5%, 96.0%, 100%, and 100%, respectively. Specificity of LRP for P. vivax, P. falciparum, P. malariae, and P. ovale was 100%, 99.3%, 100%, and 100%, respectively. Cohen's Kappa coefficients between LRP and CFX96 for detecting P. vivax, P. falciparum, P. malariae, and P. ovale were 0.96, 0.98, 1.00, and 1.00, respectively. Significant difference was not observed between the results of LRP and conventional RT-PCR and microscopic examination. A time required to amplify DNAs using LRP and conventional RT-PCR was 27 min and 86 min, respectively. LRP amplified DNAs 2 times more fast than conventional RT-PCR due to the faster heat transfer. Therefore, LRP could be employed as a useful tool for detecting malaria parasites in clinical laboratories.

• 한국동물위생학회지
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• 제31권4호
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• pp.485-492
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• 2008

Salmonella enterica serovars Gallinarum(SG, causative agent of fowl typhoid) and S Pullorum(SP, causative agent of pullorum disease) are very important bacterial pathogens in poultry industry. They share some common antigenic properties though the characteristics of outbreaks are quite different. To discriminate between SG and SP, we developed two rapid diagnostic methods, allele-specific real-time PCR and 3'-tailed PCR over 2 single nucleotide polymorphisms ($237^{th}\;and\;598^{th}$). In both methods, $237^{th}$ allele was found to be a good target for differential diagnosis, while $598^{th}$ allele produced some non-specific reactions.