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1.
Foodborne Pathog Dis ; 19(2): 126-135, 2022 02.
Article in English | MEDLINE | ID: mdl-34726510

ABSTRACT

Diarrheagenic Escherichia coli (DEC) causes diarrheal symptoms in humans. The comprehensive detection of DEC from feces using SYBR Green real-time PCR assay requires multiple runs. Moreover, PCR screening can have discrepancies related to the conformance between the results from PCR screening and culturing. We aimed to develop a real-time PCR for the comprehensive testing of DEC for diagnostic support that can be used in any general laboratory and proposed its effective utilization. We tested specificity for the designed primer sets using 100 strains. Moreover, screening and isolation of DEC were performed using the proposed multiplex real-time PCR system for 308 fecal samples collected from 37 food poisoning incidences that occurred in Gifu Prefecture, Japan from 2017 to 2019. Furthermore, the factor of discrepant results between PCR screening and culturing was analyzed by quantifying the number of DEC cell and whole E. coli cell using real-time PCR for 47 PCR screening-positive fecal samples. The results obtained from the developed multiplex real-time PCR system were in 99% concordance with those from the conventional techniques. A total of 49 fecal samples were detected with virulence genes for the screening. Of the samples which were positive with virulence genes by PCR screening, 38.3% could not be detected from the strain for bacterial culture. We found that the culturing positive samples were significantly high in numbers for the DEC cells, but no significant difference was noted in the whole E. coli cells with culturing negative samples. The multiplex real-time PCR developed in this study was found to be rapid and practical for DEC testing. The PCR screening for DEC using this method can provide rapid information toward the diagnostic support of DEC infection.


Subject(s)
Escherichia coli Infections , Foodborne Diseases , Diarrhea/microbiology , Escherichia coli/genetics , Escherichia coli Infections/diagnosis , Escherichia coli Infections/epidemiology , Escherichia coli Infections/microbiology , Feces/microbiology , Foodborne Diseases/diagnosis , Humans , Multiplex Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/methods
2.
Jpn J Infect Dis ; 74(6): 587-591, 2021 Nov 22.
Article in English | MEDLINE | ID: mdl-33952767

ABSTRACT

To investigate the molecular epidemiological characteristics of Mycobacterium tuberculosis strains collected from patients in Gifu Prefecture, Japan, 483 M. tuberculosis clinical isolates were analyzed using Japan Anti-Tuberculosis Association (JATA) 18-variable number tandem repeats (VNTR) between 2015 and 2019. To evaluate the lineage of M. tuberculosis strains, JATA18-VNTR profiles were applied to a maximum a posteriori method. The results revealed that the ancient Beijing subfamily, accounting for 57.3% (277/483) of the strains was the most prevalent M. tuberculosis strain. Furthermore, 18 clusters (GC-1-18) were found by minimum spanning tree analysis. The proportion of clustering strains was 9.9% (48/483), and epidemiological links to these clusters were unclear without GC-6 and GC-18. Meanwhile, interestingly, VNTR profiles of GC-7-9 and GC-14 were indistinguishable from the regional epidemic strains of Nagoya City, which has a strong socioeconomic relationship with Gifu Prefecture, but did not match the nationwide epidemic strains. This study suggests that coordinated analyses within the prefectures with strong socioeconomic relationships are important.


Subject(s)
Molecular Typing/methods , Mycobacterium tuberculosis/genetics , Tuberculosis/ethnology , Adult , Female , Genotype , Humans , Japan/epidemiology , Male , Middle Aged , Minisatellite Repeats , Mycobacterium tuberculosis/isolation & purification , Prevalence , Tuberculosis/diagnosis
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