Limits...
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

HRM curve profiles of the ten most frequent spa types performed on an LC 480 real-time instrument.t032, t022, t041, t2203, t001, t002, t003, t5712, t008, t190 double spiked with genomic DNA of spa types t030 and t003.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4358892&req=5

pone.0116713.g003: HRM curve profiles of the ten most frequent spa types performed on an LC 480 real-time instrument.t032, t022, t041, t2203, t001, t002, t003, t5712, t008, t190 double spiked with genomic DNA of spa types t030 and t003.

Mentions: To improve the performance of the assay and to achieve a separation of spa types with closely related or indistinguishable melting curve profiles, reaction mixtures were individually spiked with an equal amount of genomic DNA from six arbitrarily chosen spa types (t001, t003, t008, t030, t190 and t3220). Single spiking with spa type t003 or spa type t030 yielded the best results but did not allow separation of all spa types (data not shown). Spiking with DNA from spa type t003 failed to resolve spa type t008 and spa type t068 curve profiles and spiking with DNA from spa type t030 failed to resolve spa type t001, t002 and t003 curve profiles more clearly (data not shown). Adding equal amounts of genomic DNA of spa types t003 and t030 to the reaction mixture resulted in optimal assay performance that allowed clear differentiation of all spa types, that had displayed indistinguishable melting curve profiles in preliminary experiments [t003 and t023, t041 and t2945 (data not shown), t068, t3220, t008, t030, t037, t919, t190 and t011] (Fig. 2). For a better comparison and visualisation of the spiking effect on melting curve profiles spiked and unspiked samples were analyzed on the same plate (Fig. 2). Spiking resulted in better discrimination of the ten most frequent spa types and of t001, t002 and t003 (Fig. 3).


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)

HRM curve profiles of the ten most frequent spa types performed on an LC 480 real-time instrument.t032, t022, t041, t2203, t001, t002, t003, t5712, t008, t190 double spiked with genomic DNA of spa types t030 and t003.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0116713.g003: HRM curve profiles of the ten most frequent spa types performed on an LC 480 real-time instrument.t032, t022, t041, t2203, t001, t002, t003, t5712, t008, t190 double spiked with genomic DNA of spa types t030 and t003.
Mentions: To improve the performance of the assay and to achieve a separation of spa types with closely related or indistinguishable melting curve profiles, reaction mixtures were individually spiked with an equal amount of genomic DNA from six arbitrarily chosen spa types (t001, t003, t008, t030, t190 and t3220). Single spiking with spa type t003 or spa type t030 yielded the best results but did not allow separation of all spa types (data not shown). Spiking with DNA from spa type t003 failed to resolve spa type t008 and spa type t068 curve profiles and spiking with DNA from spa type t030 failed to resolve spa type t001, t002 and t003 curve profiles more clearly (data not shown). Adding equal amounts of genomic DNA of spa types t003 and t030 to the reaction mixture resulted in optimal assay performance that allowed clear differentiation of all spa types, that had displayed indistinguishable melting curve profiles in preliminary experiments [t003 and t023, t041 and t2945 (data not shown), t068, t3220, t008, t030, t037, t919, t190 and t011] (Fig. 2). For a better comparison and visualisation of the spiking effect on melting curve profiles spiked and unspiked samples were analyzed on the same plate (Fig. 2). Spiking resulted in better discrimination of the ten most frequent spa types and of t001, t002 and t003 (Fig. 3).

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