One-Step Triplex High-Resolution Melting Analysis for Rapid Identification and Simultaneous Subtyping of Frequently Isolated Salmonella Serovars

Zeinzinger, Josef; Pietzka, Ariane; Stöger, Anna; Kornschober, Christian; Kunert, Renate; Allerberger, Franz; Mach, Robert L.; Ruppitsch, Werner

doi:10.1128/aem.07668-11

Cited by 22 publications

(20 citation statements)

References 26 publications

(27 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The HRM has been effectively used for species identification of five human hookworm species, variation scanning for differentiation, ie. between “cattle type” and “sheep type” of Mycobacterium avium [21] or genotyping of human hookworm [22], Pseudomonas savastanoi [23], noroviruses [24], Salmonella serovars [25], and the identification of recent and non-recent HIV infections [26]. To date, HRM has not yet been applied with real-time PCR for detection and genotyping of opportunistic foodborne pathogens Cronobacter spp.…”

Section: Introductionmentioning

confidence: 99%

Rapid Detection and Simultaneous Genotyping of Cronobacter spp. (formerly Enterobacter sakazakii) in Powdered Infant Formula Using Real-time PCR and High Resolution Melting (HRM) Analysis

Cai

ZhouXi

et al. 2013

PLoS ONE

View full text Add to dashboard Cite

Cronobacter spp. is an emerging pathogen that causes meningitis, sepsis, bacteremia, and necrotizing enterocolitis in neonates and children. The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis targeting the OmpA gene for the specific detection and rapid identification of Cronobacter spp. (formerly Enterobacter sakazakii) in powdered infant formula. Eleven Cronobacter field isolates and 25 reference strains were examined using one pair of primers, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit of 102 CFU/ml without pre-enrichment, and highly concordant (100%) when compared with ISO-IDF 22964 in 89 actual samples. The method performed for Cronobacter spp. detection was less than 24 h, drastically shortened, compared to several days using standard culturing method, it is probe-free and reduces a risk of PCR carryover. Moreover, all Cronobacter strains examined in this study were genotyped into two species according to their HRM profiles. The established method should provide a molecular tool for direct detection and simultaneous genotyping of Cronobacter spp. in powdered infant formula.

show abstract

Section: Introductionmentioning

confidence: 99%

Rapid Detection and Simultaneous Genotyping of Cronobacter spp. (formerly Enterobacter sakazakii) in Powdered Infant Formula Using Real-time PCR and High Resolution Melting (HRM) Analysis

Cai

ZhouXi

et al. 2013

PLoS ONE

View full text Add to dashboard Cite

show abstract

“…Monitoring fluorescence of saturating dye enables the identification of Tm where the Tm of DNA duplex is the temperature at which the normalized fluorescence is 50%. HRM presents a simple, low-cost, and rapid method to scan for known and unknown mutations [19], including a large number of samples. Recent real-time PCR instruments and intercalating dyes applied at saturating concentrations have enabled closed-tube discrimination of very subtle sequence changes in PCR amplicons [20].…”

Section: Introductionmentioning

confidence: 99%

A High Resolution DNA Melting Curve Analysis for the Rapid and Efficient Molecular Diagnostics of Extended Spectrum β-Lactamase Determinants from Foodborne Escherichia coli

Njage

Buys

2020

Microorganisms

View full text Add to dashboard Cite

The accurate identification of Extended-Spectrum β-Lactamase (ESBL) genes in Gram-negative bacteria is necessary for surveillance and epidemiological studies of transmission through foods. We report a novel rapid, cheap, and accurate closed tube molecular diagnostic tool based on two multiplex HRM protocols for analysis of the predominant ESBL families encountered in foods. The first multiplex PCR assay targeted blaCTX-M including phylogenetic groups 1 (CTX-M-1-15, including CTX-M-1, CTX-M-3 and CTX-M-15), 2 (CTX-M-2), and 9 (CTX-M-9-14, including CTX-M-9 and CTX-M-14). The second assay involved blaTEM /bla CTX-M /blaSHV, including TEM variants (TEM-1 and TEM-2), SHV-1-56 (SHV-1, SHV-2 and SHV-56), and CTX-M-8-41 (CTX-M-8, CTX-M-25, CTX-M-26 and CTX-M-39 to CTX-M-41). The individual melting curves were differentiated by a temperature shift according to the type of ESBL gene. The specificity and sensitivity of the first assay were 100% and 98%, respectively. For the second assay, the specificity and sensitivity were 87% and 89%, respectively. The detection of ESBL variants or mutations in existing genes was also demonstrated by the subtyping of a variant of the CTXM-1-15. The HRM is a potential tool for the rapid detection of present β-lactamase genes and their characterization in a highly sensitive, closed-tube, inexpensive method that is applicable in high throughput studies.

show abstract

“…Despite numerous other attempts, such as fatty acid composition studies (5), restriction fragment length polymorphism (RFLP) analysis (15,16), large-sequence polymorphism analysis (17), multilocus sequence analysis (4), or multispacer sequence typing (18), some of which had promising results but were tested on only two to five isolates (4,16,17) (20).…”

mentioning

confidence: 99%

Molecular Identification of Mycobacterium avium subsp. silvaticum by Duplex High-Resolution Melt Analysis and Subspecies-Specific Real-Time PCR

2015

View full text Add to dashboard Cite

bAccurate identification of mycobacterial species and subspecies is essential to evaluate their significance and to perform epidemiological studies. The subspecies of Mycobacterium avium have different attributes but coincide in their zoonotic potential. Our knowledge about M. avium subsp. silvaticum is limited, since its identification is uncertain. Mycobacterium avium subsp. avium and M. avium subsp. silvaticum can be discriminated from each other based only on phenotypic characteristics, as they have almost identical genome sequences. Here we describe the development of a diagnostic method which enables the molecular identification of M. avium subsp. silvaticum and discrimination from M. avium subsp. avium based on genomic differences in a duplex high-resolution melt and M. avium subsp. silvaticum-specific mismatch real-time PCR. The developed assay was tested on reference strains and 199 field isolates, which were analyzed by phenotypic methods previously. This assay not only identified all 63 M. avium subsp. silvaticum and 138 M. avium subsp. avium strains correctly but also enabled the detection of mixed M. avium subsp. avium-M. avium subsp. silvaticum cultures. This is the first time that such a large panel of strains has been analyzed, and we also report the first isolation of M. avium subsp. silvaticum from red fox, red deer, wild boar, cattle, and badger. This assay is reliable, rapid, simple, inexpensive, and robust. It eliminates the long-existing problem of ambiguous phenotypic identification and opens up the possibility for detailed and comprehensive strain studies.T he species Mycobacterium avium is divided into three subspecies according to the currently valid taxonomical classification: Mycobacterium avium subsp. paratuberculosis, the etiological agent of Johne's disease; Mycobacterium avium subsp. avium, the pathogen of avian tuberculosis; and Mycobacterium avium subsp. silvaticum (1). In addition, the designation "Mycobacterium avium subsp. hominissuis" has been proposed for human/porcine-type M. avium isolates (2). M. avium subsp. silvaticum, previously called "wood pigeon Mycobacterium," was assigned to the Mycobacterium avium complex (MAC) in 1990 (3). Besides wood pigeons, it was isolated from crane (3), penguin (4), roe deer (5), and hazel hen (5). The zoonotic potential of MAC members is renowned (6), but less is known about M. avium subsp. silvaticum; hence, the detection and study of this subspecies are hindered by its unreliable and ambiguous identification.Accurate identification of mycobacterial species and subspecies is essential for evaluation of their significance, pathogenicity, and epidemiology. The four subspecies of M. avium differ greatly in their host range, growth potential, and environmental occurrence (4). Three subspecies can be easily differentiated by molecular biological methods thanks to distinct molecular differences. IS900 is the specific insertion sequence (IS) of M. avium subsp. paratuberculosis. "M. avium subsp. hominissuis" and M. avium subsp. avium both harbo...

show abstract

One-Step Triplex High-Resolution Melting Analysis for Rapid Identification and Simultaneous Subtyping of Frequently Isolated Salmonella Serovars

Cited by 22 publications

References 26 publications

Rapid Detection and Simultaneous Genotyping of Cronobacter spp. (formerly Enterobacter sakazakii) in Powdered Infant Formula Using Real-time PCR and High Resolution Melting (HRM) Analysis

Rapid Detection and Simultaneous Genotyping of Cronobacter spp. (formerly Enterobacter sakazakii) in Powdered Infant Formula Using Real-time PCR and High Resolution Melting (HRM) Analysis

A High Resolution DNA Melting Curve Analysis for the Rapid and Efficient Molecular Diagnostics of Extended Spectrum β-Lactamase Determinants from Foodborne Escherichia coli

Molecular Identification of Mycobacterium avium subsp. silvaticum by Duplex High-Resolution Melt Analysis and Subspecies-Specific Real-Time PCR

Contact Info

Product

Resources

About