• Korean Journal of Veterinary Research
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• v.38 no.1
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• pp.77-83
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• 1998

돼지 생식기호흡기증후군(porcine reproductive and respiratory syndrome, PRRS) 바이러스가 웅돈에 감염되었을 경우에는 정충의 기형 등 정액의 질 저하와 더불어 정액에 바이러스가 함유되어 있어 종부시 모돈에 바이러스를 전파하는 것으로 알려져 있다. 감염된 웅돈의 정액으로 인공수정을 실시할 경우 농장의 모돈 전체에 순식간에 바이러스를 전파하여 막대한 피해를 유발할 가능성이 높다. 따라서 감염웅돈을 신속히 검색하여 격리함으로써 피해를 사전에 방지해야 하며, 이를 위해서 웅돈의 감염여부를 신속히 확진하는 진단법의 개발이 필요한 실정이다. PRRS의 진단을 위해서는 바이러스학적인 진단법으로는 바이러스 분리동정, 혈청학적인 방법으로 바이러스 분리동정이 필수적이나 검사시간이 많이 소요되고, 분리동정 자체가 까다로운 단점이 있다. 본 연구에서는 웅돈의 정액내에서 PRRSV 바이러스에 대한 유전자를 RT-PCR법으로 증폭하는 방법을 개발하였으며, 진단의 민감도를 높이기 위하여 Nested PCR법으로 재확인 할 경우, 바이러스의 역가가 $1TCID_{50}$만 함유되어 있어도 진단이 가능한 조건을 확립하였다. 이 방법을 이용할 경우 웅돈의 정액시료에 대한 PRRS 바이러스 감염여부를 신속, 정확하게 검색하여 감염웅돈을 통한 PRRS바이러스의 전파를 미리 차단할 수 있으므로 PRRS방제에 효과적으로 이용될 것으로 사료된다.

• Korean Journal of Veterinary Service
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• v.37 no.1
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• pp.29-33
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• 2014

This study aimed at finding a fast, sensitive, and efficient protocol for molecular identification of intracellular protozoa Toxoplasma (T.) gondii. For molecular detection of T. gondii, we developed a polymerase chain reaction coupled with dot blot hybridization assay (PCR/DBH). For DBH analysis, the amplified DNA of T. gondii tachyzoite was labeled by incorporation of digoxigenin. The DBH assay alone was capable of detecting down to $1{\times}10^4$ pg of T. gondii genomic DNA. The PCR alone was capable of detecting down to $1{\times}10^3$ pg of T. gondii genomic DNA, whereas the PCR/DBH assay was capable of detecting down to $1{\times}10^2$ pg of T. gondii genomic DNA, indicating that sensitivity of the PCR/DBH method was approximately 10 to 100 times higher than PCR or DBH alone. Our PCR/DBH assay will be useful for confirming the presence of T. gondii on the samples and differentiating T. gondii infection from other intracellular protozoa infections.

• Journal of Microbiology
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• v.38 no.2
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• pp.105-108
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• 2000

Oligonucleotide primers amplifying a 344 bp fragment on the integrin-like protein alpha-INT1p gene (${\alpha}$INT1) of Candida albicans were synthesized for screenign of C. albicans from clinicalsamples by the polymerase chain reaction (PCR). The PCR specifically amplified DNA from C. albicans and none from any other Candida, fungal, or human DNA in standard used here. The PCR assay showed that the primers (LH1 and LH2) were specific for 26 isolates of C. albicans from clinical smaples, whereas the positive fragment, 344 bp, was not amplified from 15 clinical isolates including 14 other medically important Candida species and an isolate of Saccharomyces cerevisiae. PCR was conducted on the urine samples of 20 patients and 4 samples were C. albicans positive. The detection limit of the PCR assay for C. albicans was shown to be approximately 10 cells/ml saline. The PCR system using 344 bp ${\alpha}$INT1 as a target is more specific and rapid than the conventional culture method, and the sensitive detection method is applicable to clinical diagnosis of C. albicans infections.

• Parasites, Hosts and Diseases
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• v.49 no.3
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• pp.281-284
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• 2011

Amebiasis is a protozoan disease caused by Entamoeba histolytica and a potential health threat in areas where sanitation and hygiene are inappropriate. Highly sensitive PCR methods for detection of E. histolytica in clinical and environmental samples are extremely useful to control amebiasis and to promote public health. The present study compared several primer sets for small subunit (SSU) rDNA and histone genes of E. histolytica cysts. A 246 bp of the SSU rDNA gene of pure cysts contained in phosphate-buffered saline (PBS) and in stool samples was successfully amplified by nested PCR, using the 1,147-246 bp primer set, of the primary PCR products which were pre-amplified using the 1,147 bp primer as the template. The detection limit of the nested PCR using the 1,147-246 primer set was 10 cysts in both groups (PBS and stool samples). The PCR to detect histone gene showed negative results. We propose that the nested PCR technique to detect SSU rDNA can be used as a highly sensitive genetic method to detect E. histolytica cysts in stool samples.

• Food Science and Biotechnology
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• v.17 no.6
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• pp.1171-1177
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• 2008

This study was undertaken to classify Enterobacter sakazakii isolates from 13 powdered infant formula products, 25 powdered weaning diet products, and 33 weaning diet ingredients on polymerse chain reaction (PCR) methods. The numbers of the isolates from 1 powdered infant formula product, 7 powdered weaning diet products, and 6 weaning diet ingredients were 1, 14, and 8, respectively. The contaminated ingredients were 1 rice powder, 2 millet powders, 2 vegetable powders, and 1 fruit and vegetable premix. PCR with the primer of repetitive extragenic palindromic element (REP-PCR) and random amplification of polymorphic DNA(RAPD) were effective in discriminating among the isolates, but tRNA-PCR and PCR with the primer of l6S-23S internal transcribed spacer (ITS-PCR) were not. Some of E. sakazakii isolates from vegetable powders, fruit and vegetable premix, and millets powders were classified into the clonal groups based on the DNA patterns in the REP-PCR and RAPD analysis. A close genetic relationship among the isolates from some of the powdered weaning diet products and the rice powder was also detected in the cluster analysis based on the DNA patterns in RAPD.

• Food Science and Biotechnology
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• v.14 no.3
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• pp.387-391
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• 2005

Competitive polymerase chain reaction (cPCR) was used to develop a direct enumeration method of Listeria monocytogenes in pork meat. Pork meat was artificially inoculated with L. monocytogenes and DNA was extracted using guanidine thiocyanate-phenol-chloroform and subjected to PCR amplification. Sixteen primer sets for L. monocytogenes hlyA gene were tested for sensitive detection and the DG69/DG74 primer set was selected. The detection limit achieved with this primer set was as low as 860 colony-forming units (cfu) per 0.1 g of pork meat. When the samples were cultured at $30^{\circ}C$ for 16 hr in Brain Heart Infusion (BHI) medium, even a single bacterium could be detected with this primer set by PCR. For cPCR, the hlyA gene, which features a 148 bp-deletion, was cloned in the pGEM-4Z vector. A known amount of competitor DNA which has the same primer binding sites was co-amplified with L. monocytogenes total DNA from the artificially inoculated pork meat. The cell-number determined by cPCR was approximately equal to cfu from the Most Probable Number (MPN) method. The whole procedure took only 5 hr.

• Parasites, Hosts and Diseases
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• v.60 no.4
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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.

• Journal of Microbiology and Biotechnology
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• v.33 no.11
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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.

• Korean Journal of Ecology and Environment
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• v.37 no.1 s.106
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• pp.122-129
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• 2004

In an effort to develop the biomarker for monitoring the contamination of xenoestrogen in the freshwater environment of Korea, reverse transcription-polymerasechain reaction (RT-PCR) analysis of vitellogenin (VTG) gene expression was optimized in Hearisarsus Iaseo, Based on the homology of the VTG cDNA sequences between the common carp and zebra fish, a set of PCR primers for VTG mRNA amplification for H; labo was designed. VTG mRNA level in livers from female and male fishes was analyzed by RT-PCR following single injection of 17 beta estradiol($E_2$ 10 mg $kg^{-1}$ B.W.). As an internal control, beta actin mRNA was amplified. One us of total liver RNA was subjected to RT-PCR. In female the amount of PCR productof VfC gradually increased in the range from 16 to 34 cycles of amplification. On the contrary, in control male, PCR product first detected at 32 cycles of amplification and linearly increased up to 40 cycles of amplification. In $E_2$ injected male liver, the VTC mRNA level was similar to that in the female. Taken together, this result suggests that liver of male H. labo expresses minute amount of VTG mRNA which are2-l6 equivalent of female and that induction of VTG mRNA occurs in male liver after estrogen treatment. In conclusion, the optimized protocol for RT-PCR analysis of VTG mRNA expression in liver of male H. labo will provide the environmental monitoring method for the xenoestrogen contamination in the rivers in Korea.

• Journal of Microbiology and Biotechnology
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• v.27 no.4
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• pp.816-824
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• 2017

Human noroviruses are widespread and contagious viruses causing nonbacterial gastroenteritis. Real-time reverse transcription quantitative PCR (real-time RT-qPCR) is currently the gold standard for the sensitive and accurate detection of these pathogens and serves as a critical tool in outbreak prevention and control. Different surveillance teams, however, may use different assays, and variability in specimen conditions may lead to disagreement in results. Furthermore, the norovirus genome is highly variable and continuously evolving. These issues necessitate the re-examination of the real-time RT-qPCR's robustness in the context of accurate detection as well as the investigation of practical strategies to enhance assay performance. Four widely referenced real-time RT-qPCR assays (Assays A-D) were simultaneously performed to evaluate characteristics such as PCR efficiency, detection limit, and sensitivity and specificity with RT-PCR, and to assess the most accurate method for detecting norovirus genogroups I and II. Overall, Assay D was evaluated to be the most precise and accurate assay in this study. A ZEN internal quencher, which decreases nonspecific fluorescence during the PCR, was added to Assay D's probe, which further improved the assay performance. This study compared several detection assays for noroviruses, and an improvement strategy based on such comparisons provided useful characterizations of a highly optimized real-time RT-qPCR assay for norovirus detection.