• Journal of Microbiology and Biotechnology
• /
• v.21 no.1
• /
• pp.89-92
• /
• 2011

The routine identification and differentiation of Brucella species is a time-consuming and labor-intensive process, which frequently places personnel at risk of laboratory-acquired infection. Here, we describe the development of a rapid multiplex PCR assay for the confirmation of presumptive Brucella isolates. The assay was able to identify and differentiate major human pathogens, namely B. abortus, B. melitensis, and B. suis, in a single test of less than an hour and a half.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.6
• /
• pp.862-868
• /
• 2014

Certain Escherichia coli (E. coli) strains have the ability to cause diarrheal disease. Five types of diarrheagenic E. coli have been identified, including EHEC, ETEC, EPEC, EAEC, and EIEC. To detect these five diarrheagenic types rapidly, we developed a one-step multiplex PCR (MP-PCR) assay using nine primer pairs to amplify nine virulence genes specific to the different virotypes, with each group being represented (i.e., stx1 and stx2 for EHEC, lt, sth, and stp for ETEC, eaeA and bfpA for EPEC, aggR for EAEC, and ipaH for EIEC). The PCR primers were constructed using MultAlin. The sensitivity and specificity of the constructed multiplex PCR primers were measured using DNA isolated from diarrheagenic E. coli strains representing each group. The limits of detection were as follows: $5{\times}10^1CFU/ml$ for EHEC, $5{\times}10^3CFU/ml$ for ETEC expressing lt and sth, $5{\times}10^4CFU/ml$ for ETEC expressing stp, $5{\times}10^2CFU/ml$ for EPEC, $5{\times}10^4CFU/ml$ for EAEC, and $5{\times}10^2CFU/ml$ for EIEC. To confirm the specificity, C. jejuni, C. perfringens, S. Typhimurium, V. parahaemolyticus, L. monocytogenes, Y. enterocolitica, B. cereus, and S. aureus were used as negative controls, and no amplification was obtained for these. Moreover, this kit was validated using 100 fecal samples from patients with diarrhea and 150 diarrheagenic E. coli strains isolated in Korea. In conclusion, the multiplex PCR assay developed in this study is very useful for the rapid and specific detection of five diarrheagenic E. coli types. This single-step assay will be useful as a rapid and economical method, as it reduces the cost and time required for the identification of diarrheagenic E. coli.

• The Plant Pathology Journal
• /
• v.22 no.2
• /
• pp.168-173
• /
• 2006

To develop the diagnostic method for the viral infection in apple, the partial genes corresponding to the N-terminal region of RNA polymerase of Apple stem grooving virus (ASGV) and coat protein of Apple chlorotic leaf spot virus (ACLSV) were characterized from the infected apple cultivars in Korea. Based on the nucleotide sequences of the characterized partial genes, the virus gene-specific primers were designed for the detection of ASGV and ACLSV infected in species of Malus. The RT-PCR using the primers for the genes of ASGV and ACLSV successfully gave rise to 404 and 566 bp DNA fragments, respectively. Using those viral gene-specific primers, the multiplex RT-PCR assays were also established to diagnose the mixed infection by ASGV and ACLSV simultaneously. Furthermore, the control primers, which have to be included for the RT-PCR as an internal control, were designed using the nucleotide sequence of the gene encoding elongation factor $1{\alpha}(EF1{\alpha})$. This multiplex RT-PCR including the control primers provides more reliable, rapid and sensitive assay for the detection of ASGV and ACLSV infected in Korean apple cultivars.

• Korean Journal of Veterinary Research
• /
• v.45 no.3
• /
• pp.351-357
• /
• 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.

• Korean Journal of Veterinary Research
• /
• v.52 no.1
• /
• pp.39-43
• /
• 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.

• Biomedical Science Letters
• /
• v.4 no.1
• /
• pp.43-56
• /
• 1998

A multiplex PCR method was designed by employing primers specific for the eaeA gene, conserved sequences of Shiga-like toxins (SLT-I.II), and the 60-MDa plasmid of enterohemorrhagic E. coli (EHEC) O157:H7 strain. A set of six synthetic oligonucleotide primers derived from sequences of the SLT-I.II, eaeA, and 60-MDa plasmid genes of E. coli O157:H7 were used in a multiplex PCR amplification procedure to detect these genes in the same enteric pathogens. In two enterohemorrhagic E. coli O157:H7 (ATCC 35150, ATCC 43894) reference strains, PCR products of 317bps (eaeA), 228bps (SLT-I.II), and 167bps (60-MDa plasmid) were successfully amplified simultaneously in a single reaction. However, the specific PCR products were not amplified in control strains of other enteric bacteria. The sensitivity of the multiplex PCR assay for detection of the SLT-I.II, eaeA, and 60-MDa plasmid genes of E. coli O157:H7 was found to be 2.5$\times$10$^{6}$ of bacteria in diarrheal stool to amplify all three bands. The multiplex PCR technology will allow large-scale screening of many clinical specimens or contaminated foods, and will be a very useful method for the detection of a wide range of microorganisms present in the environment, including EHEC O157:H7 in various types of specimens. The multiplex PCR assay has the potential to be used as a specific and rapid method for clinical diagnosis of disease caused by EHEC O157:H7.

• Food Science of Animal Resources
• /
• v.40 no.4
• /
• pp.628-635
• /
• 2020

The objective of this study was to detect three non-halal meat products consisted of dog, pork, and rat species in meatball using novel multiplex-PCR with 12S rRNA gene as target sites. A total of 33 self-made meatballs were used, and they were grouped into eleven types of meatball based on meat species origin contained in the meatballs. Each type consisted of three meatballs. Extraction of genomic DNA from the meatballs was used as a DNA template for simplex-, duplex-, and multiplex-PCR processes. The result of simplex-PCR, duplex-PCR, and multiplex-PCR showed that the 12S rRNA primer gene successfully amplified DNA for each species bovine, dog, pig, and rat, which are respectively indicated by 155, 244, 357, and 491 bp of DNA bands. In addition, multiplex-PCR with 12S rRNA gene primers can be uniquely and accurately used for detection bovine, dog, pig, and rat species on beef meatball in one reaction.

• The Plant Pathology Journal
• /
• v.30 no.1
• /
• pp.96-101
• /
• 2014

The molecular features of Pseudomonas syringae pv. actinidiae strains isolated in Korea were compared with strains isolated in Japan and Italy. Sequencing of eight P. syringae pv. actinidiae and three P. syringae pv. theae strains revealed a total of 44 single nucleotide polymorphisms across 4,818 bp of the concatenated alignment of nine genes. A multiplex PCR assay was developed for the detection of P. syringae pv. actinidiae and for the specific detection of recent haplotype strains other than strains isolated since the 1980s in Korea. The primer pair, designated as TacF and TacR, specifically amplified a 545-bp fragment with the genomic DNA of new haplotype of P. syringae pv. actinidiae strains. A multiplex PCR conducted with the TacF/TacR primer pair and the universal primer pair for all P. syringae pv. actinidiae strains can be simultaneously applied for the detection of P. syringae pv. actinidiae and for the differentiation of new haplotype strains.

• Journal of Microbiology
• /
• v.44 no.1
• /
• pp.92-97
• /
• 2006

We developed an mPCR assay for the simultaneous detection, in one tube, of Escherichia coli O157:H7, Salmonella spp., Staphylococcus aureus and Listeria monocytogenes using species-specific primers. The mPCR employed the E. coli O157:H7 specific primer Stx2A, Salmonella spp. specific primer Its, S. aureus specific primer Cap8A-B and L. monocytogenes specific primer Hly. Amplification with these primers produced products of 553, 312, 405 and 210 bp, respectively. All PCR products were easily detected by agarose gel electrophoresis, and the sequences of the specific amplicons assessed. Potential pathogenic bacteria, in laboratory-prepared and four commercially available kimchi products, were using this mPCR assay, and the amplicons cloned and sequenced. The results correlated exactly with sequences derived for amplicons obtained during preliminry tests with known organisms. The sensitivity of the assay was determined for the purified pathogen DNAs from four strains. The mPCR detected pathogen DNA at concentrations ranging from approximately 0.45 to $0.05\;pM/{\mu}l$. Thus, this mPCR assay may allow for the rapid, reliable and cost-effective identification of four potentially pathogens present in the mixed bacterial communities of commercially available kimchi.

• Korean Journal of Veterinary Research
• /
• v.52 no.2
• /
• pp.75-82
• /
• 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.