Rapid Detection and Identification of Clinically Important Bacteria by High-Resolution Melting Analysis after Broad-Range Ribosomal RNA Real-Time PCR

Cheng, Ju Chien; Huang, Chien‐Ling; Lin, Chih-Kuang; Chen, Chi Ching; Chang, Yu Han; Chang, Shy Shin; Tseng, Ching Ping

doi:10.1373/clinchem.2006.069286

Cited by 145 publications

(104 citation statements)

References 30 publications

(22 reference statements)

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“…Also, high-resolution melting (HRM) curve analysis without using a fluorescence hybridization probe has been introduced recently as a rapid technique for genotyping and mutation scanning (Gundry et al, 2003). The introduction of new fluorescent dyes, such as LCGreen I and SYTO 9 green (Krypuy et al, 2006;Monis et al, 2005), has brought further improvement to HRM curve analysis for clinical (Reed & Wittwer, 2004;Wittwer et al, 2003;Zhou et al, 2005) and/or epidemiological (Cheng et al, 2006;Odell et al, 2005;Robinson et al, 2006) investigations.…”

Section: Introductionmentioning

confidence: 99%

Classification of Mycoplasma synoviae strains using single-strand conformation polymorphism and high-resolution melting-curve analysis of the vlhA gene single-copy region

et al. 2007

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Mycoplasma synoviae is an economically important pathogen of poultry worldwide, causing respiratory infection and synovitis in chickens and turkeys. Identification of M. synoviae isolates is of critical importance, particularly in countries in which poultry flocks are vaccinated with the live attenuated M. synoviae strain MS-H. Using oligonucleotide primers complementary to the single-copy conserved 59 end of the variable lipoprotein and haemagglutinin gene (vlhA), amplicons of~400 bp were generated from 35 different M. synoviae strains/isolates from chickens and subjected to mutation scanning analysis. Analysis of the amplicons by single-strand conformation polymorphism (SSCP) revealed 10 distinct profiles (A-J). Sequencing of the amplicons representing these profiles revealed that each profile related to a unique sequence, some differing from each other by only one base-pair substitution. Comparative high-resolution melting (HRM) curve analysis of the amplicons using SYTO 9 green fluorescent dye also displayed profiles which were concordant with the same 10 SSCP profiles (A-J) and their sequences. For both mutation detection methods, the Australian M. synoviae strains represented one of the A, B, C or D profiles, while the USA strains represented one of the E, F, G, H, I or J profiles. The results presented in this study show that the PCR-based SSCP or HRM curve analyses of vlhA provide high-resolution mutation detection tools for the detection and identification of M. synoviae strains. In particular, the HRM curve analysis is a rapid and effective technique which can be performed in a single test tube in less than 2 h.

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Section: Introductionmentioning

confidence: 99%

Classification of Mycoplasma synoviae strains using single-strand conformation polymorphism and high-resolution melting-curve analysis of the vlhA gene single-copy region

et al. 2007

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“…However, these methods require separate DNA amplification procedures and sophisticated equipment that are not always available on a routine basis. Recently, real-time PCR combined with high-resolution melting analysis has also been explored for rapid bacteria identification (25 ). In all, 25 clinically important bacterial species could be identified after LightCycler real-time PCR amplification of the 16S rRNA gene in the presence of LCGreen I fluorescent dye followed by high-resolution melting analysis of the PCR products with the HR-1 instrument.…”

Section: Discussionmentioning

confidence: 99%

Identification of 8 Foodborne Pathogens by Multicolor Combinational Probe Coding Technology in a Single Real-Time PCR

Huang

2007

Clinical Chemistry

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Background: Real-time PCR assays have been widely used for detecting foodborne pathogens but have been much less frequently applied in species identification, mainly because of the low number of species they can distinguish in 1 reaction. The present study used a new probe coding/labeling strategy, termed multicolor combinational probe coding (MCPC), to increase the number of targets that can be distinguished in a single real-time PCR for rapid and reliable species identification. Methods: With MCPC, 8 pairs of species-specific tagged primers, 1 pair of universal primers, and 8 unilabeled or mix-labeled molecular beacon probes were included in a single reaction tube. Real-time PCR was performed, and the identity of each of the 8 pathogens was determined by amplification profile comparison. The method was validated via blind assessment of 118 bacterial strains, including clinical isolates and isolates from food products. Results: The blind test with 118 samples gave no falsepositive or -negative results for the target genes. The template DNA suitable for MCPC analysis was simply prepared by heating lysis, and the total PCR analysis was finished within 2.5 h, excluding template preparation. Conclusions: MCPC is suitable for rapid and reliable identification of foodborne pathogens at the species level.

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“…In the field of broad-range bacterial species identification, Klaschik et al 22 were the first to develop a PCR and melting analysis assay using 2 probes for identification of 17 common bacterial species. In 2006, Cheng et al 23 differentiated and identified 46 bacterial culture colonies representing 25 clinically important species using a LightCycler real-time PCR instrument for amplification of the 16S rRNA gene in the presence of the LC Green I fluorescent dye and a HR-1 melting instrument for melting profiles, without using any probe. Simplicity and smartness of this assay that promised rapid and economic bacterial species identification in future was instantly recognized and commented on in Editorial 24 .…”

Section: Melting Analysis For Species Identification Purposesmentioning

confidence: 99%

The Potential of High Resolution Melting Analysis (Hrma) to Streamline, Facilitate and Enrich Routine Diagnostics in Medical Microbiology

Ruskova¹,

Raclavsky²

2011

BIOMED PAP

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Background. Routine medical microbiology diagnostics relies on conventional cultivation followed by phenotypic techniques for identification of pathogenic bacteria and fungi. This is not only due to tradition and economy but also because it provides pure culture needed for antibiotic susceptibility testing. This review focuses on the potential of High Resolution Melting Analysis (HRMA) of double-stranded DNA for future routine medical microbiology.Methods and Results. Search of MEDLINE database for publications showing the advantages of HRMA in routine medical microbiology for identification, strain typing and further characterization of pathogenic bacteria and fungi in particular. The results show increasing numbers of newly-developed and more tailor-made assays in this field. For microbiologists unfamiliar with technical aspects of HRMA, we also provide insight into the technique from the perspective of microbial characterization.Conclusions. We can anticipate that the routine availability of HRMA in medical microbiology laboratories will provide a strong stimulus to this field. This is already envisioned by the growing number of medical microbiology applications published recently. The speed, power, convenience and cost effectiveness of this technology virtually predestine that it will advance genetic characterization of microbes and streamline, facilitate and enrich diagnostics in routine medical microbiology without interfering with the proven advantages of conventional cultivation.

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Rapid Detection and Identification of Clinically Important Bacteria by High-Resolution Melting Analysis after Broad-Range Ribosomal RNA Real-Time PCR

Cited by 145 publications

References 30 publications

Classification of Mycoplasma synoviae strains using single-strand conformation polymorphism and high-resolution melting-curve analysis of the vlhA gene single-copy region

Classification of Mycoplasma synoviae strains using single-strand conformation polymorphism and high-resolution melting-curve analysis of the vlhA gene single-copy region

Identification of 8 Foodborne Pathogens by Multicolor Combinational Probe Coding Technology in a Single Real-Time PCR

The Potential of High Resolution Melting Analysis (Hrma) to Streamline, Facilitate and Enrich Routine Diagnostics in Medical Microbiology

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