• 대한수의학회지
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• 제52권1호
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• pp.39-43
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• 2012

A multiplex PCR was developed for the simultaneous detection and differentiation of Mycoplasma (M.) hyopneumoniae and M. hyorhinis in clinical samples. Improved sensitivity is advantage of this technique over the previously reported multiplex assay. It was capable of detecting as little as 125 fg genomic DNA from M. hyopneumoniae and 62.5 fg genomic DNA from M. hyorhinis. Application of this multiplex PCR method to field isolates showed that M. hyopneumoniae and M. hyorhinis were present in 29% (107 of 370) of lung specimens and no mycoplasmas were detected in 56% (208 of 370) of the slaughtered pigs' lungs. At the farm level, M. hyopneumoniae and M. hyorhinis were detected in 34 of 36 (94.4%) randomly selected farms. We conclude that this assay would prove itself a value tool for monitoring these mycoplasmal infections and both M. hyopneumoniae and M. hyorhinis have been widely spread in swine herds of Korea.

• Clinical and Experimental Pediatrics
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• 제55권11호
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• pp.424-429
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• 2012

Purpose: Methods for quick and reliable detection of Streptococcus pneumoniae are needed for the diagnosis of pneumococcal disease and vaccine studies. This study aimed to show that sequential multiplex polymerase chain reaction (PCR) is more efficient than conventional culture in achieving S. pneumoniae -positive results. Methods: Nasopharyngeal (NP) secretions were obtained from 842 pediatric patients admitted with lower respiratory infections at Severance Children's Hospital in Korea between March 2009 and June 2010. For identification and serotype determination of pneumococci from the NP secretions, the secretions were evaluated via multiplex PCR technique with 35 serotype-specific primers arranged in 8 multiplex PCR sets and conventional bacteriological culture technique. Results: Among the results for 793 samples that underwent both bacterial culture and PCR analysis for pneumococcal detection, 153 (19.3%) results obtained by PCR and 81 (10.2%) results obtained by conventional culture technique were positive for S. pneumoniae. The predominant serotypes observed, in order of decreasing frequency, were 19A (23%), 6A/B (16%), 19F (11%), 15B/C (5%), 15A (5%), and 11A (4%); further, 26% of the isolates were non-typeable. Conclusion: As opposed to conventional bacteriological tests, PCR analysis can accurately and rapidly identify pneumococcal serotypes.

• Journal of Microbiology and Biotechnology
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• 제17권3호
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• pp.490-495
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• 2007

Lactic acid bacteria (LAB) are beneficial for the gastrointestinal tract and reinforce immunity in human health. Recently, many functional products using the lactic acid bacteria have been developed. Among these LAB, Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum, and Bifidobacterium bifidum are frequently used for probiotic products. In order to monitor these LAB in commercial probiotic products, a multiplex PCR method was developed. We designed four species-specific primer pairs for multiplex PCR from the 16S rRNA, 16S-23S rRNA intergenic spacer region, and 23S rRNA genes in Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum, and Bifidobacterium bifidum. Using these primer pairs, 4 different LAB were detected with high specificity in functional foods. We suggest that the multiplex PCR method developed in this study would be an efficient tool for simple, rapid, and reliable identification of LAB used as probiotic strains.

• Journal of Microbiology and Biotechnology
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• 제28권2호
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• pp.210-217
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• 2018

A one-step multiplex reverse transcription PCR (RT-PCR) method comprising six primer sets (for the detection of norovirus GI and GII, hepatitis A virus, rotavirus, and astrovirus) was developed to simultaneously detect four kinds of pathogenic viruses. The size of the PCR products for norovirus GI and GII, hepatitis A virus (VP3/VP1 and P2A regions), rotavirus, and astrovirus were 330, 164, 244, 198, 629, and 449 bp, respectively. The RT-PCR with the six primer sets showed specificity for the pathogenic viruses. The detection limit of the developed multiplex RT-PCR, as evaluated using serially diluted viral RNAs, was comparable to that of one-step single RT-PCR. Moreover, this multiplex RT-PCR was evaluated using food samples such as water, oysters, lettuce, and vegetable product. These food samples were artificially spiked with the four kinds of viruses in diverse combinations, and the spiked viruses in all food samples were detected successfully.

• Journal of Microbiology and Biotechnology
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• 제25권10호
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• pp.1714-1727
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• 2015

This work describes a new strategy for optimal design of Multiplex-PCR primer sequences. The process is based on the Particle Swarm Optimization-Simplex algorithm (Mult-PSOS). Diverging from previous solutions centered on heuristic tools, the Mult-PSOS is selfconfigured because it does not require the definition of the algorithm's initial search parameters. The successful performance of this method was validated in vitro using Multiplex-PCR assays. For this validation, seven gene sequences of the most prevalent bacteria implicated in urinary tract infections were taken as DNA targets. The in vitro tests confirmed the good performance of the Mult-PSOS, with respect to infectious disease diagnosis, in the rapid and efficient selection of the optimal oligonucleotide sequences for Multiplex-PCRs. The predicted sequences allowed the adequate amplification of all amplicons in a single step (with the correct amount of DNA template and primers), reducing significantly the need for trial and error experiments. In addition, owing to its independence from the initial selection of the heuristic constants, the Mult-PSOS can be employed by non-expert users in computational techniques or in primer design problems.

• Journal of Microbiology and Biotechnology
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• 제17권7호
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• pp.1177-1182
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• 2007

Bacillus cereus group bacteria share a significant degree of genetic similarity. Thus, to differentiate and identify the Bacillus cereus group efficiently, a multiplex PCR method using the gyrB and groEL genes as diagnostic markers is suggested for simultaneous detection. The assay yielded a 400 bp amplicon for the groEL gene from all the B. cereus group bacteria, and a 253 bp amplicon from B. anthracis, 475 bp amplicon from B. cereus, 299 bp amplicon from B. thuringiensis, and 604 bp amplicon from B. mycoides for the gyrB gene. No nonspecific amplicons were observed with the DNA from 29 other pathogenic bacteria. The specificity and sensitivity of the B. cereus group identification using this multiplex PCR assay were evaluated with different kinds of food samples. In conclusion, the proposed multiplex PCR is a reliable, simple, rapid, and efficient method for the simultaneous identification of B. cereus group bacteria from food samples in a single tube.

• Journal of Microbiology
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• 제39권3호
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• pp.226-228
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• 2001

Human lymphotropic herpesvirus is known to be a major pathogen associated with various diseases in bone marrow transplantation (BMT) recipients. A multiplex nested-polymerase chain reaction (PCR) method was developed for the simultaneous detection of human lymphotropic herpesviruses, including Ebstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpesvirus 6 variants A and B (HHV6-A, HHV6-B). To demonstrate the usefulness of multiplex PCR for the analysis of clinical samples, peripheral blood mononuclear cells and serum from BMT recipients were analysed. The results skewed that a clear detection could be made between EBV, HCMV and HHV-6. This multiplex PCR assay is an efficient and cost-effective approach to the analysis of large numbers of samples to determine the epidemiological importance of EBV HCMV and HHV-6.

• 한국광학회지
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• 제7권1호
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• pp.28-36
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• 1996

질소레이저의 출력을 높이고 안정성을 향상시키기 위한 방법의 하나로 8쌍으로 구성된 다극형 스파크 간격과 Blumlein 형태의 다중 평행판 축전기형 전송회로로 구성된 레이저 장치를 제작하였고 실험은 스파크 간격을 자체트리거하는 방식과 외부트리거하는 두가지 방식을 택하여 실시하였다. 그 결과 긴 레이저관에 대하여 하나의 스파크 간격이 레이저관 전체를 통하여 레이저를 균일하고 완벽하게 발진시킬수는 없음을 확인하였으며 다극형 스파크 간격을 사용할 경우에도 스파크 간격 모두가 한 몸체처럼 완벽한 역할을 하지 못함을 확인하였다. 그러나 이와 같은 복합적인 결과로 부터 다극형 스파크 간격이 레이저 출력 증가에 기여를 할 수 있음을 확인하였고 그의 설치 범위는 레이저관의 길이(또는 전송회로의 두께)의 3/4이 최적임을 알았다.

• Parasites, Hosts and Diseases
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• 제48권4호
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• pp.297-301
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• 2010

Recently, emerging waterbome protozoa, such as microsporidia, Cyclospora, and Cryptosporidium, have become a challenge to human health worldwide. Rapid, simple, and economical detection methods for these major waterborne protozoa in environmental and clinical samples are necessary to control infection and improve public health. In the present study, we developed a multiplex PCR test that is able to detect all these 3 major waterborne protozoa at the same time. Detection limits of the multiplex PCR method ranged from $10^1$ to $10^2$ oocysts or spores. The primers for microsporidia or Cryptosporidium used in this study can detect both Enterocytozoon bieneusi and Encephalitozoon intestinalis, or both Cryptosporidium hominis and Cryptosporidium paNum, respectively. Restriction enzyme digestion of PCR products with BsaBI or BsiEI makes it possible to distinguish the 2 species of microsporidia or Cryptosporidium, respectively. This simple, rapid, and cost-effective multiplex PCR method will be useful for detecting outbreaks or sporadic cases of waterborne protozoa infections.

• 한국수산과학회지
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• 제50권6호
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• pp.806-811
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• 2017

A multiplex PCR system comprising 14 microsatellite markers was developed for genotyping analysis of the sea cucumber Stichopus japonicus. A total of 286 samples were used to evaluate genetic polymorphisms and forensic parameters of the microsatellite loci. In a single PCR reaction, all 14 loci were uniformly amplified and a total of 269 alleles were identified. The AJ19024 locus had the largest number of alleles (46), and its discriminatory power and exclusion power were 0.99 and 0.76, respectively. The fewest alleles (8) were present at the Psj2575 locus, which provided the lowest discriminatory power (0.81) and exclusion power (0.20). The mean number of alleles, mean heterozygosity, mean discrimination power and mean exclusion power per locus were 19.21, 0.70, 0.93, and 0.46, respectively. The combined matching probability for the 14 loci was $9.64{\times}10^{-19}$, and the combined power of exclusion was 0.999995. Thus, the forensic parameters evaluated in the present study demonstrated the utility of our multiplex PCR system for biological tracing methods, such as individual identification and paternity testing, in the sea cucumber.