• Food Science and Biotechnology
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• 제18권3호
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• pp.694-699
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• 2009

A multiplex polymerase chain reaction (PCR) method was developed for the detection of 4 events of genetically modified (GM) soybean. The event-specific primers were designed from 4 events of GM soybean (RRS, A2704-12, DP356043-5, and MON89788). The lectin was used as an endogenous reference gene of soybean in the PCR detection. The primer pair YjLec-4-F/R producing 100 bp amplicon was used to amplify the lectin gene and no amplified product was observed in any of the 9 different plants used as templates. This multiplex PCR method allowed for the detection of event-specific targets in a genomic DNA mixture of up to 1% GM soybean mixture containing RRS, A2704-12, DP356043-5, and MON89788. In this study, 20 soybean products obtained from commercial food markets were analyzed by the multiplex PCR. As a result, 6 samples contained RRS. These results indicate that this multiplex PCR method could be a useful tool for monitoring GM soybean.

• Clinical and Experimental Pediatrics
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• 제52권12호
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• pp.1358-1363
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• 2009

목 적:호흡기 감염의 증상이 없는 소아들을 대상으로 다중 역전사중합효소연쇄반응법(multiplex reverse transcription-polymerase chain reaction; mRT-PCR)을 이용하여 비인두 상주균의 이환율을 알아보고자 하였다. 방 법:2008년 7월 25일부터 28일까지 33명의 소아들을 대상으로 비강 면봉채취법으로 검체를 채취하였으며, 이들은 검체 채취 당시 심각한 호흡기 감염의 증상이 없었다. 모아진 검체에서 DNA를 추출한 후 multiplex primer set ($Seeplex^{(R)}$ PneumoBacter ACE Detection, Seegene, Seoul, Korea)로 PCR을 진행하였다. 증폭된 반응산물은 2% agarose gel과 전기 영동 자동화 시스템인 screen tape system (Lab901, Scottland, UK)에 각각 전기 영동하여 확인하였다. 결 과:전체 33명의 소아 중 남아는 15명 여아는 18명이었으며, 나이는 3.2세에서 16.3세로 중앙값은 8.2세였다. mRT-PCR 결과 30명(90.9%)의 소아들에서 양성을 보였으며(S. pneumoniae, H. influenzae, C. pneumoniae, B. pertussis), 이들 중 13명(39.4%)에서 2가지 이상의 균이 검출되었다. 균의 종류로는 12명(36.4%)에서는 S. pneumoniae와 H. influenzae, 1명(3.0%)에서는 S. pneumoniae, H. influenzae와 C. pneumoniae이 검출되었다.. 결 론:mRT-PCR은 비인두 상주균의 동정에 있어서 민감도가 높은 방법으로 생각된다. 하지만 비인두 상주균에 대한 PCR 결과가 소아들의 임상 양상과 어느 정도 일치할지에 대해서는 더 많은 연구가 필요할 것으로 생각된다.

• Food Science and Biotechnology
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• 제17권4호
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• pp.829-832
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• 2008

A multiplex polymerase chain reaction (PCR) method was developed to simultaneously detect 4 varieties of genetically modified (GM) cotton. The event-specific primers were used to distinguish the 4 varieties of GM cotton (MON1445, MON15985, MON88913, and LLcotton25) using multiplex PCR. The acyl carrier protein 1 (Acp1) gene was used as an endogenous reference gene of cotton in the PCR detection. The primer pair Acp1-AF/AR containing a 99 bp amplicon was used to amplify the Acp1 gene and no amplified product was observed in any of the 13 different plants used as templates. This multiplex PCR method allowed for the detection of event-specific targets in a genomic DNA mixture of up to 1% GM cotton containing MON1445, MON15985, MON88913, and LLcotton25.

• Asian-Australasian Journal of Animal Sciences
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• 제27권10호
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• pp.1411-1416
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• 2014

Gender selection is important in livestock industries; for example, female calves are required in the dairy industry. Sex-sorted semen is commonly used for the production of calves of the desired gender. However, assessment of the sex ratio of the sorted semen is tedious and expensive. In this study, a rapid, cost effective and reliable method for determining the sex ratio was developed using a multiplex real-time polymerase chain reaction (PCR) assay. In this assay, the X and Y chromosome-specific markers, i.e., bovine proteolipid protein (PLP) gene and sex-determining region Y (SRY) were simultaneously quantified in a single tube. The multiplex real-time PCR assay was shown to have high amplification efficiencies (97% to 99%) comparable to the separated-tube simplex real-time PCR assay. The results obtained from both assays were not significantly different (p>0.05). The multiplex assay was validated using reference DNA of known X ratio (10%, 50%, and 90%) as templates. The measured %X in semen samples were the same within 95% confidence intervals as the expected values, i.e., >90% in X-sorted semen, <10% in Y-sorted semen and close to 50% in the unsorted semen. The multiplex real-time PCR assay as shown in this study can thus be used to assess purity of sex-sorted semen.

• Journal of Microbiology and Biotechnology
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• 제14권4호
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• pp.737-744
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• 2004

A multiplex polymerase chain reaction (PCR) was developed for the simultaneous detection and differentiation among Vairimorpha spp. and Nosema spp. and identification of Vairimorpha necatrix from Lepidoptera insects. Three sets of primers were selected from different genomic sequences to specifically amplify an 831 bp amplicon within the SSU rRNA gene, specific for both Vairimorpha spp. and Nosema spp. (MSSR primer); a 542 bp amplicon within the SSU rRNA gene, specific for Vairimorpha spp. (VSSU primer); and a 476 bp amplicon within the actin gene, specific for Vairimorpha necatrix (VNAG primer). Using the primers in conjunction with multiplex PCR, it was possible to detect Vairimorpha spp. and Nosema spp. and to differentiate between them. The sensitivity of this PCR assay was approximately 10 spores per milliliter. It is proposed that the multiplex PCR is a sensitive, specific, and rapid tool that can serve as a useful differential diagnostic tool for detecting Vairimorpha spp. and Nosema spp. in Lepidoptera insect.

• 한국동물위생학회지
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• 제32권2호
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• pp.147-153
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• 2009

Salmonella species are the most important etiologic agents of food-borne acute gastroenteritis. The most common serotypes isolated from humans are Salmonella enterica serotype Typhimurium (S. Typhimurium) and S. Enteritidis. Traditional detection methods for Salmonella are based on cultures using selective media and characterization of suspicious colonies by biochemical and serological tests. These methods are generally time-consuming and not so highly sensitive. Recently, the polymerase chain reaction (PCR) has been used as a highly sensitive, specific, and rapid test for the presence of pathogenic bacteria. In this study, a multiplex PCR (m-PCR) was used to detect S. Typhimurium and S. Enteritidis. We selected m-PCR target genes, which were the spv (virulence plasmid specific for S. Enteritidis) and sefA (S. Enteritidis fimbrial antigen) genes, fliC (H1-i antigen specific for S. Typhimurium) and a randomly cloned sequence specific for the genus Salmonella. With m-PCR, random sequence was detected from all strains of Salmonella spp, spv and sefA were detected from all strains of S. Enteritidis (100%), and fliC was detected from all strains of S. Typhimurium (100%). This assay indicate that the specificity of the m-PCR make them potentially valuable tools for detection of S. Typhimurium and S. Enteritidis.

• 한국식품위생안전성학회지
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• 제25권3호
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• pp.278-280
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• 2010

This study was aimed to develop a novel qualitative multiplex polymerase chain reaction (PCR) for simultaneous detection of genetically modified (GM) soy and maize within a single reaction. The specific primers designed to detect four respective GM events (A2704-12, MON88017, Bt11, and MON863) were included in the tetraplex PCR system. Each of PCR products for four GM events could be distinguished by agarose gel based on their different lengths. The specificity and reproducibility of this multiplex PCR were evaluated. This multiplex PCR consistently amplified only a fragment corresponding to a specific inserted gene in each of the four GM events and also amplified all four of the PCR products in the simulated GM mixture. These results indicate that this multiplex PCR method could be an effective qualitative detection method for screening GM soy and maize in a single reaction.

• 대한수의학회지
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• 제39권4호
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• pp.806-810
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• 1999

A multiplex-polymerase chain reaction (PCR) assay was used to detect staphylococcal enterotoxin production by 12 strains of Staphylococcus aureus isolated from clinical specimens. To evaluate the efficacy and/or sensitivity of this method, the results were compared to those obtained with the reversed passive latex agglutination kit (SET-RPLA, Denka Seiken, Japan). Of 10 strains positive by PCR were positive by RPLA but two strains, representing high sensitivity of the former method. Enterotoxin B was the most prevalent by the two methods. The kappa index between the two methods was 0.826, indicating a higher agreement and fully reliable for use. These results would suggest that sensitive, inexpensive, and relatively rapid multiplex-PCR technique may be an effective means for the detection of staphylococcal enterotoxin genes as an alternative to traditional methods such as kits or immunological methods, which depend upon the amount of enterotoxin produced.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.150.1-150
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• 2003

A single-step multiplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed for the simultaneous detection of five cucurbit-infecting viruses： cucumber mosaic virus (CMV), watermelon mosaic virus 2 (WMV2), zucchini yellow mosaic virus (ZYMV), cucumber green mottle mosaic virus (CGMMV), and kyuri green mottle mosaic virus (KGMMV). The multiplex RT-PCR provides a simple and rapid method for detecting various viruses in cucurbit plants, which will help diagnose many cucurbit plants at a time.

• 한국식품영양과학회지
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• 제39권4호
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• pp.595-601
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• 2010

본 연구는 국내 주요 식중독 원인균인 Staphylococcus aureus, Salmonella enterica subsp., Vibrio parahaemolyticus를 동시에 검출 및 동정할 수 있는 simultaneous multiplex PCR방법을 개발하고자 하였다. S. aureus의 23s rRNA 유전자(482 bp), V. Parahaemolyticus의 toxR 유전자(368 bp), S. enterica subsp.의 invA 유전자(284 bp)를 특이적으로 검출 및 동정할 수 있는 3개 primer set 즉, STA-5F/STA-5R, ToxR-F/ToxR-R, 139/141을 구축하였으며, 그 결과 정제되어진 각 식중독 원인균의 genomic DNA를 template로 하여 세 균주 모두 10 pg까지 다중동시검출이 가능하였다. 생균수(CFU)와 상응되는 검출한계 결과로써 $10^1\sim10^2$ CFU/reaction의 검출한계를 보였으며 이는 즉, S. aureus $6.0\times10^4$ CFU/mL, S. enterica subsp. $9.5\times10^4$ CFU/mL, V. parahaemolyticus $6.1\times10^5$ CFU/mL의 검출한계를 나타내었다. 균체회수부터 agarose gel 상에서 검출 및 동정까지 3~4 hr의 시간 소요로 single tube 반응으로 세 식중독 원인균의 다중동시검출이 가능하였다. 또한 추가적인 연구를 통하여 세 식중독 원인균주의 검출을 위한 향상된 민감도를 가지는 multiplex PCR법 및 real time PCR을 이용한 다중동시검출법 개발을 위한 기초자료로서 활용 가능할 것이라 사료된다.