Multiplex Polymerase Chain Reaction을 이용한 Extended-Spectrum β-Lactamase 생성 Klebsiella pneumoniae 균주의 검출

양병선 ( Byoung Seon Yang ) 대한임상검사과학회 2006 대한임상검사과학회지(KJCLS) Vol.38 No.3

The production of extended-spectrum β-lactamases (ESBLS) is the main mechanism of bacterial resistance to third-generation cephalosporins and monobactams, whose prevalence varies depending on the different geographical areas. In the last years it has increased notably to the point of being considered a health problem of great importance. The characterization of the ESBLs producing Klebsiella penumoniae strains present in clinical isolates is time-consuming. I describe here the development of a new system, which consists of a multiplex PCR. I found 51 K. pneumoniae strains to be presumptive strains ESBLs producers by clinical and laboratory standards institute (CLSI) guidelines. The double disc synergy test showed 47 positive K. pneumoniae, which were K. pneumoniae isolates. All ESBLs producing K. pneumoniae strains were resistant to antibiotic amikacin, gentamicin and ciprofloxacin. By multiplex PCR analysis, blaTEM gene in 17 strains 44 blaSHV genes and blaCTX genes in 33 strains were identified. In this study, the multiplex polymerase chain reaction (PCR) assay was a good method to detect and differentiate ESBLs producing K. penumoniae strains in clinical isolates.

Multiplex-Polymerase Chain Reaction을 이용한 대마의 조기 성판별 Sequence Characterized Amplified Region 마커 개발

김경주,고은지,신예림,김창혁,권태형,한준희,임학태,류병렬,임정대 한국약용작물학회 2023 한국약용작물학회지 Vol.31 No.3

Background: Cannabis are typically dioecious, with female and male reproductive parts located on separate plants. Female cannabis plants are preferred for the production of cannabinoids, ter- penes, and other valuable compounds synthesized within the female floral tissue. Female and male plants look identical during seedling and vegetative phases, and only after transition to flowering phase can male, female, and monoecious plants be distinguished. Early identification of the sex of cannabis plant can conserve time and money by avoiding the undesired sex of the plant. The objec- tive of the present study was to provide a sex determination method for cannabis plants using the sequence characterized amplified region (SCAR) marker, and set multiplex-polymerase chain reac- tion (PCR) conditions for more accurate early sex determination of cannabis to overcome the dis- advantages of SCAR markers. Methods and Results: Cannabis seeds were collected and cultivated for several purposes. Leaf samples were harvested and stored during vegetative growth phase. In silico mapping of three male-specific sequences (MADC3, MADC4, and MADC6) revealed through random amplified polymorphic DNA, showed that MADC3 has a very similar sequence with the female cannabis genome. Primers were produced based on the sequences (SCAR1, SCAR2, and SCAR3, respec- tively) and subjected to gradient PCR. Only SCAR3 produced a male-specific band with an anneal- ing temperature of over 61.4 ℃. PCR was performed on cannabis plants produced for several purposes by mixing SCAR3 and internal standard 3 (IS3). Result showed that all cannabis plants produced an IS3 band of 197 bp, but only male cannabis plants produced a SCAR3 band of 118 bp. Conclusions: Multiplex-PCR primer produced a male-specific band of 119 bp in cannabis bred for several purposes, and the presence of the 197 bp cannabis common band confirmed that PCR pro- ceeded normally for all samples. The multiplex-PCR primer is greatly beneficial because the IS3 band is a specific sequence found only in cannabis, making it possible to differentiate cannabis from other plants in addition to determining the sex from with a single PCR run.

Multiplex Polymerase Chain Reaction을 이용한 당귀 종 판별

김용상,박혁주,이동희,김현규 한국약용작물학회 2018 한국약용작물학회지 Vol.26 No.1

Background: Angelica gigas, A. sinensis, and A. acutiloba are commercially important in the herbal medicine market, and among them, A. gigas has the highest economic value and price. However, their similar morphological traits are often used for fraud. Despite their importance in herbal medicine, recognition of the differences between Angelica species is currently inadequate. Methods and Results: A multiplex polymerase chain reaction (PCR) method was developed for direct detection and identification of A. gigas, A. sinensis, and A. acutiloba. The gene for the distinction of species was targeted at ITS in the nucleus and trnC-petN gene in chloroplasts. The optimized multiplex PCR in the present study utilized each Angelica species-specific primer pairs. Each primer pair yielded products of 229 base pairs (bp) for A. gigas, 53 bp for A. sinensis, 170 bp for A. acutiloba. Additionally non-specific PCR products were not detected in similar species by species-specific primers. Conclusions: In the present study, a multiplex-PCR assay, successfully assessed the authenticity of Angelica species (A. gigas, A. sinensis, and A. acutiloba). and whole genome amplification (WGA) was performed after DNA extraction to identify, the species in the product. The detection method of raw materials developed in the present study could be applied to herbal medicine and health functional food management.

Multiplex PCR Detection for 3 Events of Genetically Modified Maize, DAS-59122-7, TC6275, and MIR604

Ji-Hye Ahn,Jae-Hwan Kim,Su-Youn Kim,Woo-Young Lee,Sun-Hee Park,Hae-Yeong Kim 한국식품과학회 2008 Food Science and Biotechnology Vol.17 No.3

A multiplex polymerase chain reaction (PCR) method was developed to simultaneously detect 3 events of genetically modified (GM) maize. The event-specific primers were used to discriminate the following 3 events of GM maize (DAS-59122-7, TC6275, and MIR604) using multiplex PCR method. The zein gene was used as an endogenous maize reference gene in the multiplex PCR detection. The primer pair Zein-F/R producing a 99 bp amplicon was used to amplify the zein gene. The primer JI-Das-F1/R1 for DAS-59122-7, JI-TC6275-F3/R3 for TC6275, and JI-MIR F1/R1 for MIR604 yielded an amplicon of 130, 162, and 197 bp, respectively. The detection limit of multiplex PCR was 1% for DAS-59122-7, TC6275, and MIR604 for one reaction.

Epidemiological analysis of Escherichia coli O157 : H7 by pulsed-field gel electrophoresis and multiplex polymerase chain reaction

Jung, Byeong-yeal,Jung, Suk-chan,Cho, Dong-hee,Kim, Jong-yeom,Kim, Bong-hwan The Korean Society of Veterinary Science 1999 大韓獸醫學會誌 Vol.39 No.2

Twenty three strains of Escherichia (E) coli O157 : H7 isolated from Korea, Japan, USA were analyzed by pulsed-field gel electrophoresis (PFGE) of XbaI-digested chromosomal DNA and multiplex polymerase chain reaction. Various PFGE patterns of E. coli O157 : H7 were found on the same farm. Most of the E, coli O157 : H7 strains had shiga-like toxin (slt) II gene only (43.5%) or both slt I and slt II genes(30.4%). eaeA gene was highly conserved in the E. coli O157 : H7. There was no correlation between PFGE and slt gene patterns. The results indicate that various genotypes of E. coli O157 : H7 have spread throughout the country and genomic DNA patterns generated by PFGE are highly specific for different strains and have significant value in epidemiologic investigations of infectious disease outbreaks.

원인불명 무정자증 환자에서 Y 염색체 장완내 Azoospermia Factor 부위의 미세결실

백재승,김기동,김수웅 大韓泌尿器科學會 1998 Korean Journal of Urology Vol.39 No.9

장구균의 vancomycin 내성 유전자와 종 특이유전자의 검출을 위한 Multiplex polymerase chain reaction 개발

조윤상,이희수,김종만,안종삼,류판동,박용호,유한상,이문한,Cho, Yun-Sang,Lee, Hee-Soo,Kim, Jong-Man,Ahn, Jong-Sam,Ryu, Pan-Dong,Park, Yong-Ho,Yoo, Han-Sang,Lee, Mun-Han 대한수의학회 2003 大韓獸醫學會誌 Vol.43 No.1

A multiplex PCR assay, which allows simultaneous detection of vancomycin resistant genotypes and Enterococcus species-specific genes, was developed. Vancomycin resistant enterococci (VRE) from chickens and humans could be detected for vanA, vanB, vanC-1, vanC-2, $ddl_{E.faecium}$ and $ddl_{E.faecalis}$ by multiplex PCR. Eight isolates of VRE from humans (n=11) had $ddl_{E.faecium}$ and vanA, and 3 isolates of the VRE had $ddl_{E.faecium}$ and vanB. One isolate of VRE from chickens (n=6) had $ddl_{E.faecium}$ and vanA, and 5 isolates of the VRE had only vanA. E. faecium, E. faecalis, E. gallinarum and E. casseliflavus were also confirmed for the species-specific gene by multiplex PCR. This multiplex PCR could detect E. faecium, E. faecalis, E. gallinarum, E. casseliflavus, vanA, vanB, vanC-1 and vanC-2, simultaneously. The PCR assay established in the present study can be an alternative to time-consuming biochemical tests and antibiotic susceptibility tests of Enterococcus spp.

급성 설사 환자에서 대변 다중중합효소연쇄반응 검사의 유용성

김서현,김유선,김승혁,윤원의,명희준,문정섭,황동희 대한소화기학회 2022 대한소화기학회지 Vol.79 No.3

Background/Aims: There is a recent increase in the use of stool multiplex PCR assay-based diagnostic tests in patients with acute diarrhea. We used multiplex PCR assays to analyze the distribution of diarrhea-causing bacteria and viruses, as well as the clinical features of patients with acute diarrhea. Methods: We retrospectively reviewed stool specimens of inpatients complaining of acute diarrhea from October 2018 to July 2020. The stool specimens had been tested for bacteria and viruses using multiplex PCR assays. Results: A total of 414 stool specimens from 346 patients were tested, and 152 pathogens were detected in 131 stool samples (131/414, 31.6%). Co-infection was detected in 20 patients (20/346, 5.8%). The common pathogens detected as causes of acute diarrhea, including co-infection, were Clostridium perfringens (34.9%), Clostridioides difficile (19.7%), and Campylobacter spp. (18.4%). The average age of patients with multiplex PCR-positive tests was lower than those with multiplex PCR-negative tests (p=0.001). In patients with suspected C. difficile infection (CDI), the RT-PCR for toxin gene assay was performed in 370 stool samples, 35 of which were positive (9.5%). Furthermore, 16 of the 35 samples were positive on the multiplex PCR assay (45.7%). Conclusions: The multiplex PCR assay revealed that C. perfringens was the most common diarrhea-causing pathogen. In addition, in patients with suspected CDI, the multiplex PCR assay alone was insufficiently sensitive to detect pathogens and a conventional CDI test was additionally required.

Outcomes and clinical relevance of stool multiplex bacterial polymerase chain reaction in patients with acute diarrhea: single center experience

Won Gun Kwack,Yun Jeong Lim,Ki Hwan Kwon,Jae Woo Chung,Jin Young Oh 대한내과학회 2020 The Korean Journal of Internal Medicine Vol.35 No.2

Background/Aims: Diagnostic stool multiplex polymerase chain reaction (PCR) testing has attracted considerable interest, because of its high sensitivity, short turnaround time, and ability to detect multiple organisms simultaneously. This study investigates the clinical usefulness of a stool multiplex bacterial PCR in patients with acute diarrhea. Methods: We retrospectively evaluated the stool multiplex bacterial PCR results, clinical parameters, and clinical courses of patients hospitalized because of acute diarrhea between August 2014 and November 2016. Results: A total of 725 patients (male, 372; mean age, 30.9 ± 29.3 years) underwent stool multiplex bacterial PCR. A total of 243 pathogens were detected in 226 patients. The detection rate of multiplex PCR testing was higher than that of stool culture (32.7% vs. 3.3%, p < 0.01). Severe symptoms of acute diarrhea (bloody diarrhea, frequent diarrhea) and prescribed empirical antibiotics were significantly more common in the positive multiplex PCR group (p = 0.02, p < 0.01, p < 0.01, respectively). However, mean durations of hospital stay were similar in the 2 groups according to the multiplex PCR results (p = 0.32). In addition, Campylobacter spp., which was the most commonly detected pathogen (97/243, 39.9%), was significantly associated with frequent diarrhea and prescribed empirical antibiotics (p < 0.01), but not with duration of hospital stay (p = 0.09). Conclusions: We concluded that stool multiplex bacterial PCR might be a useful tool for identifying bacterial etiology in patients with acute diarrhea, especially in those with Campylobacter spp. infection.

Genetic Monitoring of Multi-Functional Plant Growth Promoting Rhizobacteria Bacillus subtilis AH18 and Bacillus Licheniformis K11 by Multiplex and Real-Time Polymerase Chain Reaction in a Pepper Farming Field

( Jong Hui Lim ),( Chang Hwan Ahn ),( Hee Young Jeong ),( Yo Hwan Kim ),( Sang Dal Kim ) 한국응용생명화학회 2011 Applied Biological Chemistry (Appl Biol Chem) Vol.54 No.2

The plant growth promoting rhizobacteria (PGPR) strains Bacillus subtilis AH18 and Bacillus licheniformis K11 were selected as biocontrol agents for the suppression of phytophthora blight caused by Phytophthora capsici. A genetic monitoring method was developed utilizing multiplex and real-time polymerase chain reaction (PCR) in a pepper farming soil. 2,3-Dihydro-2,3-dihydroxy benzoate dehydrogenase of a key siderophore synthesis enzyme (sid), auxin efflux carrier gene (aec), and cellulase gene (cel) of B. subtilis AH18 were used as genetic methods to monitor the presence and concentration of the inoculated microbial agents. Monitoring of B. licheniformis K11 was carried out by amplification of a cellulase gene (celK), siderophore synthase gene (dhbF), and iturin synthase A gene (ituA). B. subtilis AH18 and B. lichenifomis K11 could be detected upto 20 days by multiplex PCR in pepper farming soil. B. subtilis AH18 sid and three Bacillus lichenifomis K11 genes could be detected upto week 5 by real-time PCR in the natural unsterilized soil. P. capsici pathogen became nearly undetectable after 20 days biocontrol treatment in the field soil. These results could lead to the development of select PGRPs as useful microbial agents for the organic farming of peppers.