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Improved protocol for rapid identification of certain spa types using high resolution melting curve analysis.

Mayerhofer B, Stöger A, Pietzka AT, Fernandez HL, Prewein B, Sorschag S, Kunert R, Allerberger F, Ruppitsch W - PLoS ONE (2015)

Bottom Line: The ten most frequent spa types were identified from all samples and on all instruments with 100% specificity and 100% sensitivity.Compared to classical spa typing by sequence analysis, this gene scanning assay is faster, cheaper and can be performed in a single closed tube assay format.Therefore it is an optimal screening tool to detect the most frequent endemic spa types and to exclude non-endemic spa types within a hospital.

View Article: PubMed Central - PubMed

Affiliation: University of Applied Sciences Wiener Neustadt, Department of Biomedical Analytics, Wiener Neustadt, Austria; Austrian Agency for Health and Food Safety, Institute of Medical Microbiology and Hygiene, Vienna, Austria.

ABSTRACT
Methicillin-resistant Staphylococcus aureus is one of the most significant pathogens associated with health care. For efficient surveillance, control and outbreak investigation, S. aureus typing is essential. A high resolution melting curve analysis was developed and evaluated for rapid identification of the most frequent spa types found in an Austrian hospital consortium covering 2,435 beds. Among 557 methicillin-resistant Staphylococcus aureus isolates 38 different spa types were identified by sequence analysis of the hypervariable region X of the protein A gene (spa). Identification of spa types through their characteristic high resolution melting curve profiles was considerably improved by double spiking with genomic DNA from spa type t030 and spa type t003 and allowed unambiguous and fast identification of the ten most frequent spa types t001 (58%), t003 (12%), t190 (9%), t041 (5%), t022 (2%), t032 (2%), t008 (2%), t002 (1%), t5712 (1%) and t2203 (1%), representing 93% of all isolates within this hospital consortium. The performance of the assay was evaluated by testing samples with unknown spa types from the daily routine and by testing three different high resolution melting curve analysis real-time PCR instruments. The ten most frequent spa types were identified from all samples and on all instruments with 100% specificity and 100% sensitivity. Compared to classical spa typing by sequence analysis, this gene scanning assay is faster, cheaper and can be performed in a single closed tube assay format. Therefore it is an optimal screening tool to detect the most frequent endemic spa types and to exclude non-endemic spa types within a hospital.

No MeSH data available.


Related in: MedlinePlus

Unspiked HRM curve profiles of the ten most frequent spa types.t032, t022, t041, t2203, t001, t002, t003, t5712, t008, t190 on an LC 480 real-time instrument.
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pone.0116713.g001: Unspiked HRM curve profiles of the ten most frequent spa types.t032, t022, t041, t2203, t001, t002, t003, t5712, t008, t190 on an LC 480 real-time instrument.

Mentions: An HRM assay for spa scanning has been developed using 20 MRSA isolates of the ten most frequent spa types (t001 (n = 2), t003 (n = 2), t190 (n = 2), t041 (n = 2), t022 (n = 2), t032 (n = 2), t008 (n = 2), t002 (n = 2), t5712 (n = 2) and t2203 (n = 2) (with t032, t003, t002 and t008 the four most frequent (32.6%) international types included) out of an arbitrary collection of 212 MRSA isolates (Fig. 1). HRM curve analysis yielded distinct and characteristic melting curve profiles for each tested spa type on an LC 480 instrument, but spa types t001, t002 and t003 displayed closely related melting curve profiles (Fig. 1).


Improved protocol for rapid identification of certain spa types using high resolution melting curve analysis.

Mayerhofer B, Stöger A, Pietzka AT, Fernandez HL, Prewein B, Sorschag S, Kunert R, Allerberger F, Ruppitsch W - PLoS ONE (2015)

Unspiked HRM curve profiles of the ten most frequent spa types.t032, t022, t041, t2203, t001, t002, t003, t5712, t008, t190 on an LC 480 real-time instrument.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4358892&req=5

pone.0116713.g001: Unspiked HRM curve profiles of the ten most frequent spa types.t032, t022, t041, t2203, t001, t002, t003, t5712, t008, t190 on an LC 480 real-time instrument.
Mentions: An HRM assay for spa scanning has been developed using 20 MRSA isolates of the ten most frequent spa types (t001 (n = 2), t003 (n = 2), t190 (n = 2), t041 (n = 2), t022 (n = 2), t032 (n = 2), t008 (n = 2), t002 (n = 2), t5712 (n = 2) and t2203 (n = 2) (with t032, t003, t002 and t008 the four most frequent (32.6%) international types included) out of an arbitrary collection of 212 MRSA isolates (Fig. 1). HRM curve analysis yielded distinct and characteristic melting curve profiles for each tested spa type on an LC 480 instrument, but spa types t001, t002 and t003 displayed closely related melting curve profiles (Fig. 1).

Bottom Line: The ten most frequent spa types were identified from all samples and on all instruments with 100% specificity and 100% sensitivity.Compared to classical spa typing by sequence analysis, this gene scanning assay is faster, cheaper and can be performed in a single closed tube assay format.Therefore it is an optimal screening tool to detect the most frequent endemic spa types and to exclude non-endemic spa types within a hospital.

View Article: PubMed Central - PubMed

Affiliation: University of Applied Sciences Wiener Neustadt, Department of Biomedical Analytics, Wiener Neustadt, Austria; Austrian Agency for Health and Food Safety, Institute of Medical Microbiology and Hygiene, Vienna, Austria.

ABSTRACT
Methicillin-resistant Staphylococcus aureus is one of the most significant pathogens associated with health care. For efficient surveillance, control and outbreak investigation, S. aureus typing is essential. A high resolution melting curve analysis was developed and evaluated for rapid identification of the most frequent spa types found in an Austrian hospital consortium covering 2,435 beds. Among 557 methicillin-resistant Staphylococcus aureus isolates 38 different spa types were identified by sequence analysis of the hypervariable region X of the protein A gene (spa). Identification of spa types through their characteristic high resolution melting curve profiles was considerably improved by double spiking with genomic DNA from spa type t030 and spa type t003 and allowed unambiguous and fast identification of the ten most frequent spa types t001 (58%), t003 (12%), t190 (9%), t041 (5%), t022 (2%), t032 (2%), t008 (2%), t002 (1%), t5712 (1%) and t2203 (1%), representing 93% of all isolates within this hospital consortium. The performance of the assay was evaluated by testing samples with unknown spa types from the daily routine and by testing three different high resolution melting curve analysis real-time PCR instruments. The ten most frequent spa types were identified from all samples and on all instruments with 100% specificity and 100% sensitivity. Compared to classical spa typing by sequence analysis, this gene scanning assay is faster, cheaper and can be performed in a single closed tube assay format. Therefore it is an optimal screening tool to detect the most frequent endemic spa types and to exclude non-endemic spa types within a hospital.

No MeSH data available.


Related in: MedlinePlus