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Original Articles
Development of a Two Triplex Real-Time Polymerase Chain Reaction for Rapid Detection of Six Carbapenemase Genes in Enterobacteriaceae
Ji Ae Choi, Song Mee Bae, Jung Wook Kim, Kwang Jun Lee
Osong Public Health Res Perspect. 2020;11(1):53-59.   Published online February 28, 2020
DOI: https://doi.org/10.24171/j.phrp.2020.11.1.08
  • 2,814 View
  • 92 Download
AbstractAbstract PDFSupplementary Material
Objectives

Carbapenem resistance is a serious clinical and public health threat. Carbapenemase can confer carbapenem resistance, and most carbapenemase genes are plasmid encoded so resistance can easily spread. In this study, we aimed to develop a novel system based on the TaqMan platform for the rapid detection of 6 clinically prevalent carbapenemase genes: Klebsiella pneumoniae carbapenemase, New Delhi metallo-β-lactamase, oxacillinase, imipenem-hydrolyzing, Verona integron-encoded metallo-β-lactamase, and Guiana extended-spectrum β-lactamase.

Methods

The triplex assay was verified by testing genomic DNA of 6 carbapenemase-producing Klebsiella pneumoniae. It was validated with a blinded panel of 310 Enterobacteriaceae isolates, including 225 carbapenemase-producers and 85 non-producers, by direct colony triplex real-time polymerase chain reaction (PCR). The real-time PCR was performed using the ABI 7500 fast instrument (Applied Biosystems, CA, USA) and specific primers for each carbapenemase target were designed to include modified peptide-nucleic acid oligonucleotides.

Results

No amplification was detected among the negative samples. The result showed 100% concordance with the genotypes previously identified. The entire assay, including DNA extraction and real-time PCR, was completed within 2 hours.

Conclusion

The newly developed triplex real-time PCR assay was useful for the rapid, accurate and simultaneous detection of 6 carbapenemase genes in Enterobacteriaceae, suggesting its potential to allow an early decision on the appropriate treatment, management, and prevention of the spread of resistant infections in hospitals.

Rapid Molecular Approach for Simultaneous Detection of Salmonella spp., Shigella spp., and Vibrio cholera
Reza Ranjbar, Ali Naghoni, Davoud Afshar, Farhad Nikkhahi, Mohsen Mohammadi
Osong Public Health Res Perspect. 2016;7(6):373-377.   Published online December 31, 2016
DOI: https://doi.org/10.1016/j.phrp.2016.10.002
  • 1,637 View
  • 20 Download
  • 7 Citations
AbstractAbstract PDF
Objectives
Gastrointestinal tract infection is still one of the serious public health problems in many geographic areas and is endemic in most countries including Iran. Early detection of the gastrointestinal tract pathogens can be extremely important. The aim of the current study was to apply a shortened time-multiplex polymerase chain reaction (PCR) for rapid and simultaneous detection of Salmonella spp., Shigella spp., and Vibrio cholera.
Methods
The standard and clinical strains of Salmonella spp., Shigella spp., and V. cholerae were used in the assay. Multiplex PCR was performed and optimized based on amplification of invA, putative integrase, and ompW genes for detecting Salmonella spp., Shigella spp., and V. cholerae, respectively. The specificity of the assay was evaluated by testing 12 different bacterial species.
Results
Only Salmonella spp., Shigella spp., and V. cholerae strains had positive results when subjected to the assay using multiplex PCR. The assay showed a high sensitivity, and no amplification products were observed in multiplex PCR with any of the other microorganisms.
Conclusion
Our study indicated that the invA, putative integrase, and ompW-based multiplex PCR assay appears to be an efficient method for rapid and simultaneous detection of Salmonella spp., Shigella spp., and V. cholerae.
Comparison of Three Different Methods for Detection of IL28 rs12979860 Polymorphisms as a Predictor of Treatment Outcome in Patients with Hepatitis C Virus
Abolfazl Fateh, Mohammadreza Aghasadeghi, Seyed D. Siadat, Farzam Vaziri, Farzin Sadeghi, Roohollah Fateh, Hossein Keyvani, Alireza H. Tasbiti, Shamsi Yari, Angila Ataei-Pirkooh, Seyed H. Monavari
Osong Public Health Res Perspect. 2016;7(2):83-89.   Published online April 30, 2016
DOI: https://doi.org/10.1016/j.phrp.2015.11.004
  • 1,627 View
  • 16 Download
  • 17 Citations
AbstractAbstract PDF
Objectives
This study aimed to evaluate the specificity, sensitivity, cost, and turn-around time of three methods of gene polymorphism analysis and to study the relationship between IL28B rs12979860 and SVR rate to pegIFN-α/RVB therapy among patients with chronic hepatitis C.
Methods
A total of 100 samples from chronic hepatitis C patients were analyzed in parallel using the three methods: direct sequencing, real-time polymerase chain reaction (PCR), amplification refractory mutation system (ARMS)-PCR.
Results
The different profiles for IL28B rs12979860 alleles (CC, CT, and TT) obtained with PCR-RFLP, ARMS-PCR, and direct sequencing were consistent among the three methods. Prevalence of rs12979860 genotypes CC, CT and TT in HCV genotype 1a was 10(19.6%), 35(68.6%), and six (11.8%), respectively, and in HCV genotype 31, it was 13(26.5%), 31(63.3%), and five (10.2%), respectively. No significant difference was seen between rs12979860 genotype and HCV genotype (p = 0.710).
Conclusion
Screening by ARMS – PCR SNOP detection represents the most efficient and reliable method to determine HCV polymorphisms in routine clinical practice.
TEM and SHV Genes in Klebsiella pneumoniae Isolated from Cockroaches and Their Antimicrobial Resistance Pattern
Abbas Doosti, Mohammad Pourabbas, Asghar Arshi, Mohammad Chehelgerdi, Hamidreza Kabiri
Osong Public Health Res Perspect. 2015;6(1):3-8.   Published online February 28, 2015
DOI: https://doi.org/10.1016/j.phrp.2014.10.011
  • 1,396 View
  • 44 Download
  • 12 Citations
AbstractAbstract PDF
Objectives
Klebsiella pneumoniae is a gram-negative rod bacterium, a known cause of community-acquired bacterial pneumonia and is an important hospital-acquired pathogen that causes severe morbidity and mortality. The aim of this study was to identify the TEM and SHV genes in K. pneumoniae isolated from cockroaches obtained from hospitals.
Methods
In this study, 250 cockroaches were collected from different hospitals in the province of Chaharmahal Va Bakhtiari, which is located in southwest Iran. The samples were examined for the presence of K. pneumoniae by plating onto a combination of culture media, and the antimicrobial susceptibility patterns of isolated K. pneumoniae from samples were evaluated using the disk diffusion test. In addition, from the culture, genomic bacterial DNA was extracted, and sequence-specific targets (TEM and SHV genes) were amplified using the polymerase chain reaction (PCR) method.
Results
Out of 250 cockroach samples collected from various hospitals, 179 samples (71.60%) were positive for K. pneumoniae. PCR reaction was performed using specific oligonucleotide primers (TEM-F, TEM-R and SHV-F, SHV-R) for the amplification of each gene, and amplified products were visualized on 1% agarose gel electrophoresis. Of all the specimens amplified by PCR in this research, 32 samples (17.87%) were positive for TEM and 15 samples (8.37%) were positive for SHV.
Conclusion
Detection of TEM and SHV genes using molecular methods and their pattern of antimicrobial resistance can provide useful information about the epidemiology of and risk factors associated with K. pneumoniae infection.
Multiplex Real-time Polymerase Chain Reaction Assays for Simultaneous Detection of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus
Jie Yeun Park, Semi Jeon, Jun Young Kim, Misun Park, Seonghan Kim
Osong Public Health Res Perspect. 2013;4(3):133-139.   Published online June 30, 2013
DOI: https://doi.org/10.1016/j.phrp.2013.04.004
  • 1,654 View
  • 20 Download
  • 15 Citations
AbstractAbstract PDF
Objectives
A multiplex real-time polymerase chain reaction (RT-PCR) method was developed for the identification of three Vibrio species: Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus.
Methods
Specific primers and probes targeting the hlyA, tlh, and vvhA genes were selected and used for multiplex real-time PCR to confirm the identification of V. cholerae, V. parahaemolyticus, and V. vulnificus, respectively. This method was applied to screen Vibrio species from environmental samples and combining it with a culture-based method, its effectiveness was evaluated in comparison with culture-based methods alone.
Results
Specific PCR fragments were obtained from isolates belonging to the target species, indicating a high specificity of this multiplex real-time PCR. No cross-reactivity with the assay was observed between the tested bacteria. The sensitivity of the multiplex real-time PCR was found to have a lower limit of 104 colony-forming units/reaction for all three Vibrio species. The combination strategy raised the isolation ratio of all three Vibrio species 1.26- to 2.75-fold.
Conclusion
This assay provides a rapid, sensitive, and specific technique to detect these three Vibrio species in the environment.
A Comparison of Subtyping Methods for Differentiating Salmonella enterica Serovar Enteritidis Isolates Obtained from Food and Human Sources
Ji-Yeon Hyeon, Jung-Whan Chon, Jun-Ho Park, Moo-Sang Kim, Young-Hee Oh, In-Soo Choi, Kun-Ho Seo
Osong Public Health Res Perspect. 2013;4(1):27-33.   Published online February 28, 2013
DOI: https://doi.org/10.1016/j.phrp.2012.12.005
  • 1,605 View
  • 20 Download
  • 20 Citations
AbstractAbstract PDF
Purpose: To evaluate the abilities of these subtyping methods, we distinguished Salmonella Enteritidis (S. Enteritidis) isolated from food products and human clinical samples between 2009 and 2010 in Seoul using five subtyping methods.
Methods
We determined the subtypes of 20 S. Enteritidis isolates from food and human sources using phage typing, antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), repetitive sequence-based PCR (rep-PCR), and multi-locus sequence typing (MLST).
Results
A total of 20 tested isolates were differentiated into six antimicrobial susceptibility patterns, three different phage types, four different PFGE profiles, seven rep-PCR patterns, and one MLST type. Food isolates were considerably more susceptible to antibiotics than human isolates. We were best able to discriminate among S. Enteritidis isolates using rep-PCR, and obtained the highest Simpson’s diversity index of 0.82, whereas other methods produced indices that were less than 0.71. PFGE pattern appeared to be more related to antimicrobial resistance and phage types of S. Enteritidis isolates than rep-PCR. MLST revealed identical alleles in all isolates at all seven loci examined, indicating no resolution.
Conclusion
The results of this study suggest that rep-PCR provided the best discriminatory power for phenotypically similar S. Enteritidis isolates of food and human origins, whereas the discriminatory ability of MLST may be problematic because of the high sequence conservation of the targeted genes.
Articles
Multiplex Real-Time Polymerase Chain Reaction-Based Method for the Rapid Detection of gyrA and parC Mutations in Quinolone-Resistant Escherichia coli and Shigella spp.
Junyoung Kim, Semi Jeon, Hyungjun Kim, Misun Park, Soobok Kim, Seonghan Kim
Osong Public Health Res Perspect. 2012;3(2):113-117.   Published online June 30, 2012
DOI: https://doi.org/10.1016/j.phrp.2012.04.004
  • 1,549 View
  • 14 Download
  • 5 Citations
AbstractAbstract PDF
Two real-time polymerase chain reaction assays were developed to detect mutations in codons 83 and 87 in gyrA and in codons 80 and 91 in parC, the main sites that causes quinolone resistance in pathogenic Escherichia coli and Shigella spp. isolates. These assays can be employed as a useful method for controlling infections caused by quinolone-resistant E coli and Shigella isolates.

PHRP : Osong Public Health and Research Perspectives