• 한국치위생학회지
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• 제17권1호
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• pp.13-25
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• 2017

Objectives: The purpose of this study was to compare colony forming unit (CFU) method and multiplex real-time polymerase chain reaction (MRT-PCR) method for accurate quantitative analysis of bacteria. Methods: We compared the CFU method and the MRT-PCR method, which are still used in Korea, for Prevotella intermedius (P. intermedius), a periodontal disease pathogen selected by MRT-PCR, and Streptococcus mutans (S. mutans), a dental caries causative organism. The subjects of this study were 30 patients who visited the C dental hospital. Results: Total microorganisms in MRT-PCR method were significantly higher in both types of bacteria (p<0.05), since DNA of dead bacteria was also analyzed. This was because the periodontal dise(-) anaerobes, and even dead bacteria contain large amounts of toxic substances called LPS in the extracellular membrane, and fimbriae and pili, which are motility structures, still remain as a strong toxic substance in periodontal tissue. Conclusions: Therefore, in terms of the total amount of bacteria found, the MRT-PCR method will be a useful technique for searching all the bacteria in the oral cavity including live bacteria, as well as sterilization.

• 대한수의학회지
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• 제45권3호
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• pp.351-357
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• 2005

Here I describe a multiplex allele specific PCR-based approach for the rapid detection between Hanwoo and Holstein meat associated with Melanocortin 1 receptor (MC1R) gene. Specific and universal oligonucleotide primers were used in combination to detect the presence of a single nucleotide polymorphism within the bovine MC1R DNA sequence. The presence of the bovine MC1R gene is indicated by the production of a single control PCR product, whilst positive samples generate an alternative smaller specific product over the same region. The mutations in MC1R104 codon revealed depending on the presence or absence of an indicative fragment amplified from the wild-type allele of this codon. As little as 0.39 ng and 1.56 ng of genomic DNA of Hanwoo and Holstein could be detected by MAS-PCR assay, respectively. This technique, which is widely used in human genetic screening, provides a reliable and sensitive result that has not been documented for the identification of bovine coat color. The MAS-PCR assay approach was proven to be useful in complementing routine beef DNA analysis for differentiation of these MC1R variants and it would facilitate the screening of deceiving sales of Holstein meat in the butcher shop.

• Journal of Microbiology and Biotechnology
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• 제19권11호
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• pp.1470-1474
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• 2009

This paper describes the development a of direct multiplex reverse transcription-nested polymerase chain reaction (PCR) method, devised for simultaneous detection and typing of influenza viruses. This method combines the direct reverse transcription reaction without RNA purification with the enhancement of sensitivity and specificity of nested PCR. The method successfully detected three major human influenza viruses: influenza virus A subtype 1 (H1N1) and subtype 3 (H3N2), and influenza B virus (B). The minimum number of virus particles (pfu/ml) necessary for detection in spiked saliva samples was 200 (H1N1), 140 (H3N2), and 4.5 (B). The method's sensitivity and simplicity will be convenient for use in clinical laboratories for the detection and subtyping of influenza and possibly other RNA viruses.

• 한국임상수의학회지
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• 제32권2호
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• pp.154-157
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• 2015

개에서 설사를 일으키는 주요 원인이 되는 장내 병원균으로 Salmonella spp., Shigella spp., Campylobacter spp., Clostridium spp.가 있다. 이들 세균은 배양으로 검출이 어렵다. 본 실험에서는 Salmonella spp., C. coli, C. jejuni, 그리고 Cl. perfringens를 신속하고 민감하게 검출할 수 있는 방법을 고안하였다. 정상견 71마리와 설사증상이 있는 66마리에서 수집한 분변 시료에서 장내 병원균의 유병률을 알아보고자 하였다. 장내 병원균은 실시간 중합효소 연쇄 반응 분석을 이용하여 검출하였다. 설사변에서 Salmonella spp., C. coli, C. jejuni, Cl. Perfringens는 정상변보다 검출률이 높았다. 개발한 다중실시간 중합효소연쇄반응은 분변시료의 병원균 존재 및 양 또는 기타 고유 서열을 확인하는데 유용하였다.

• Pediatric Infection and Vaccine
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• 제27권2호
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• pp.90-101
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• 2020

목적: 급성 위장염의 대부분의 경우 원인 병원체가 확인되지 않는다. 최근 들어 발달한 multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) 검사는 장염 병원체 검출에 도움을 줄 수 있다. 이 연구는 multiplex RT-PCR을 이용해, 소아 장염환자에서 병원체의 역학을 조사하고자 하였다. 방법: 2015년 5월부터 2018년 6월까지 대한민국 서울의 2차병원에서 급성 위장염으로 진단받은 소아 환자의 대변에서 병원체를 확인하기 위해 multiplex RT-PCR 검사를 시행하였다. 결과: 바이러스 병원체에 대한 1,366개의 대변 검체 중, 483개(35.3%)에서 1개 이상의 병원체가 분리되었다. A군 로타바이러스는 106건(7.8%)에서 확인되었으며, 양성률은 3.0% (8/263)에서 16.7% (48/288)까지 매년 증가했다(P<0.001). 노로바이러스 GII는 가장 흔한 바이러스성 병원체였고(263/1366, 19.3%), 3년간 양성률은 증가하지 않았다. 세균성 병원체에 대한 304개의 대변 검체 중 캄필로박터(32/304, 10.5%)는 가장 흔한 세균성 병원체였으며, 그 다음으로 Clostridium difficile (toxin B) (22/304, 7.2%), 살모넬라균(17/304, 5.6%)이었다. 이 균들의 양성률은 연구기간 동안 증가하지 않았다. 결론: 로타바이러스 백신 도입 이후 노로바이러스 GII가 소아 장염에서 주요한 병원체였지만, 연구기간 동안 로타바이러스 감염 환자가 증가했고, 특히 2018년에는 급증했다. 따라서 새로운 로타바이러스 균주의 등장 가능성을 포함한 추가 연구가 필요하다. 캄필로박터는 소아 세균성 장염의 주요 원인이며, 적절한 치료를 위해 이 균의 임상적 특성을 고려하고 지속적 감시가 필요하겠다.

• 대한수의학회지
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• 제52권2호
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• pp.75-82
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• 2012

We have developed and optimized a multiplex polymerase chain reaction (mPCR) for simultaneous detection of Brucella, Salmonella and Leptospira with high sensitivity and specificity. Three pairs of oligonucleotide primers were designed to specifically amplify the targeted genes of Salmonella, Leptospira and Brucella species with sizes of 521, 408 and 223 bp, respectively. The mPCR did not produce any nonspecific amplification products when tested against 15 related species of bacteria. The sensitivity of the mPCR was 100 fg for Brucella and 1 pg for both Salmonella and Leptospira species. In the field application, kidney, liver and spleen were collected from wild rats and stray cats and examined by mPCR. The high specificity and sensitivity of this mPCR assay provide a valuable tool for diagnosis and for the simultaneous and rapid detection of three zoonotic bacteria that cause disease in both humans and animals. Therefore, this assay could be a useful alternative to the conventional method of culture and single PCR for the detection of each pathogen.

• Journal of Plant Biotechnology
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• 제47권4호
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• pp.309-315
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• 2020

Advances in biotechnology have led to progress in crop genetic engineering to improve agricultural productivity. The use of genetically modified (GM) crops has increased, as have consumers' and regulators' concerns about the safety of GM crops to human health, and ecological biodiversity. As such, the identification of GM crops is a critical issue for developers and distributors, and their labeling is mandatory. Multiplex polymerase chain reaction (PCR) has been developed and its use validated for the detection and identification of GM crops in quarantine. Herein, we established a simultaneous detection method to identify four GM maize events. Event-specific primers were designed between the junction region of transgene and genome of four GM maize lines, namely 5307, DAS-40278-9, MON87460, and MON87427. To verify the efficiency and accuracy of the multiplex PCR we used specificity analysis, limit of detection evaluation, and mixed certified reference materials identification. The multiplex PCR method was applied to analyze 29 living, modified maize volunteers collected in South Korea in 2018 and 2019. We performed multiplex PCR analysis to identify events and confirmed the result by simplex PCR using each event-specific primer. As a result, rather than detecting each event individually, the simultaneous detection PCR method enabled the rapid analysis of 29 GM maize volunteers. Thus, the novel multiplex PCR method is applicable for living modified organism volunteer identification.

• 한국임상수의학회지
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• 제23권3호
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• pp.300-307
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• 2006

In the present study, methods of the reverse transcription-polymerase chain reaction(RT-PCR) were evaluated for the rapid detection and differentiation of transmissible gastroenteritis virus(TGEV), porcine epidemic diarrhea virus(PEDV) and rotavirus in piglets suffering from diarrhea. For the purposes, the PCR conditions were first confirmed for the amplification of VP7 gene of rotavirus and N gene of TGEV and PEDV using each specific primers and their annealing temperature. Multiplex RT-PCR methods were further determined to distinguish these viral infections and the results are as follows. For the specific amplification of these viral genes, the reliable PCR condition was determined as 30 cycles of reaction consisting each 1 min of denature at $94^{\circ}C$, annealing at $42^{\circ}C$ and polymerization at $72^{\circ}C$ with 1.0 mM $MgCl_2$. It was able to differentiate these viral infections in the intestines and feces of piglets suffering from diarrhea by duplex PCR for TGEV and PEDV and single PCR for rotavirus with a primer-annealing temperature of $42^{\circ}C$. When the multiplex RT-PCR were undertaken for the field samples, 17 cases of PEDV and 5 cases of rotavirus infections were differential diagnosed in a total of 92 samples of intestines and feces of the piglets with diarrhea.

• Journal of Microbiology and Biotechnology
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• 제22권8호
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• pp.1165-1169
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• 2012

In April 2009, the H1N1 pandemic influenza virus emerged as a novel influenza virus. The aim of this study was to compare the performances of several molecular assays, including conventional reverse transcription polymerase chain reaction (RT-PCR), two real-time reverse transcription (rRT)-PCRs, and two multiplex RTPCRs. A total of 381 clinical specimens were collected from patients (223 men and 158 women), and both the Seeplex RV7 assay and rRT-PCR were ordered on different specimens within one week after collection. The concordance rate for the two methods was 87% (332/381), and the discrepancy rate was 13% (49/381). The positive rates for the molecular assays studied included 93.1% for the multiplex Seeplex RV7 assay, 93.1% for conventional reverse transcription (cRT)-PCR, 89.7% for the multiplex Seeplex Flu ACE Subtyping assay, 82.8% for protocol B rRT-PCR, and 58.6% for protocol A rRT-PCR. Our results showed that the multiplex Seeplex assays and the cRT-PCR yielded higher detection rates than rRT-PCRs for detecting the influenza A (H1N1) virus. Although the multiplex Seeplex assays had the advantage of simultaneous detection of several viruses, they were time-consuming and troublesome. Our results show that, although rRT-PCR had the advantage, the detection rates of the molecular assays varied depending upon the source of the influenza A (H1N1)v virus. Our findings also suggest that rRT-PCR sometimes detected virus in extremely low abundance and thus required validation of analytical performance and clinical correlation.

• Asian Pacific Journal of Cancer Prevention
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• 제16권7호
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• pp.3003-3007
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• 2015

Objective: To explore the application of multiplex nested methylated specific polymerase chain reaction (PCR) in the early diagnosis of epithelial ovarian carcinoma (EOC). Materials and Methods: Serum and fresh tissue samples were collected from 114 EOC patients. RUNX3, TFPI2 and OPCML served as target genes. Methylation levels of tissues were assessed by multiplex nested methylated specific PCR, the results being compared with those for carcinoma antigen 125 (CA125). Results: The serum free deoxyribose nucleic acid (DNA) methylation spectrum of EOC patients was completely contained in the DNA spectrum of cancer tissues, providing an accurate reflection of tumor DNA methylation conditions. Serum levels of CA125 and free DNA methylation in the EOC group were evidently higher than those in benign lesion and control groups (p<0.05). Patients with early EOC had markedly lower serum CA125 than those with advanced EOC (p<0.05), but there was no significant difference in free DNA methylation (p>0.05). The sensitivity, specificity and positive predicative value (PPV) of multiplex nested methylated specific PCR were significantly higher for detection of all patients and those with early EOC than those for CA125 (p<0.05). In the detection of patients with advanced EOC, the PPV of CA125 detection was obviously lower than that of multiplex nested methylated specific PCR (p>0.05), but there was no significant difference in sensitivity (p>0.05). Conclusions: Serum free DNA methylation can be used as a biological marker for EOC and multiplex nested methylated specific PCR should be considered for early diagnosis since it can accurately determine tumor methylation conditions.