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Implementation and Evaluation of a Fully Automated Multiplex Real-Time PCR Assay on the BD Max Platform to Detect and Differentiate Herpesviridae from Cerebrospinal Fluids.

Köller T, Kurze D, Lange M, Scherdin M, Podbielski A, Warnke P - PLoS ONE (2016)

Bottom Line: Performance was compared to an established accredited multiplex real time PCR protocol utilizing the easyMAG and the LightCycler 480/II, both very common devices in viral molecular diagnostics.For clinical validation, 123 CSF specimens and 40 reference samples from national interlaboratory comparisons were examined with both methods, resulting in 97.6% and 100% concordance for CSF and reference samples, respectively.Workflow analyses displayed shorter test duration as well as remarkable fewer and easier preparation steps with the potential to reduce error rates occurring when manually assessing patient samples.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, Virology, and Hygiene, Rostock University Hospital, Rostock, Germany.

ABSTRACT
A fully automated multiplex real-time PCR assay--including a sample process control and a plasmid based positive control--for the detection and differentiation of herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2) and varicella-zoster virus (VZV) from cerebrospinal fluids (CSF) was developed on the BD Max platform. Performance was compared to an established accredited multiplex real time PCR protocol utilizing the easyMAG and the LightCycler 480/II, both very common devices in viral molecular diagnostics. For clinical validation, 123 CSF specimens and 40 reference samples from national interlaboratory comparisons were examined with both methods, resulting in 97.6% and 100% concordance for CSF and reference samples, respectively. Utilizing the BD Max platform revealed sensitivities of 173 (CI 95%, 88-258) copies/ml for HSV1, 171 (CI 95%, 148-194) copies/ml for HSV2 and 84 (CI 95%, 5-163) copies/ml for VZV. Cross reactivity could be excluded by checking 25 common viral, bacterial and fungal human pathogens. Workflow analyses displayed shorter test duration as well as remarkable fewer and easier preparation steps with the potential to reduce error rates occurring when manually assessing patient samples. This protocol allows for a fully automated PCR assay on the BD Max platform for the simultaneously detection of herpesviridae from CSF specimens. Singular or multiple infections due to HSV1, HSV2 and VZV can reliably be differentiated with good sensitivities. Control parameters are included within the assay, thereby rendering its suitability for current quality management requirements.

No MeSH data available.


Related in: MedlinePlus

Scheme of workflow.Abbreviations: p/p, primers/probes; PC, positive control; MM, master mix; NC, negative control.
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pone.0153991.g001: Scheme of workflow.Abbreviations: p/p, primers/probes; PC, positive control; MM, master mix; NC, negative control.

Mentions: Total time as well as hands-on time was recorded from the beginning of the procedures until the final evaluation of the results (Fig 1). Time was averaged from the work of different technicians (Fig 1). Only test runs containing the same amount of samples were incorporated into the analysis. The preparation of master mixes as well as primer/probes mixes were not included into the work flow analysis since these steps (p1, p2, p3) are inherent part of the analysis protocol.


Implementation and Evaluation of a Fully Automated Multiplex Real-Time PCR Assay on the BD Max Platform to Detect and Differentiate Herpesviridae from Cerebrospinal Fluids.

Köller T, Kurze D, Lange M, Scherdin M, Podbielski A, Warnke P - PLoS ONE (2016)

Scheme of workflow.Abbreviations: p/p, primers/probes; PC, positive control; MM, master mix; NC, negative control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836685&req=5

pone.0153991.g001: Scheme of workflow.Abbreviations: p/p, primers/probes; PC, positive control; MM, master mix; NC, negative control.
Mentions: Total time as well as hands-on time was recorded from the beginning of the procedures until the final evaluation of the results (Fig 1). Time was averaged from the work of different technicians (Fig 1). Only test runs containing the same amount of samples were incorporated into the analysis. The preparation of master mixes as well as primer/probes mixes were not included into the work flow analysis since these steps (p1, p2, p3) are inherent part of the analysis protocol.

Bottom Line: Performance was compared to an established accredited multiplex real time PCR protocol utilizing the easyMAG and the LightCycler 480/II, both very common devices in viral molecular diagnostics.For clinical validation, 123 CSF specimens and 40 reference samples from national interlaboratory comparisons were examined with both methods, resulting in 97.6% and 100% concordance for CSF and reference samples, respectively.Workflow analyses displayed shorter test duration as well as remarkable fewer and easier preparation steps with the potential to reduce error rates occurring when manually assessing patient samples.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, Virology, and Hygiene, Rostock University Hospital, Rostock, Germany.

ABSTRACT
A fully automated multiplex real-time PCR assay--including a sample process control and a plasmid based positive control--for the detection and differentiation of herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2) and varicella-zoster virus (VZV) from cerebrospinal fluids (CSF) was developed on the BD Max platform. Performance was compared to an established accredited multiplex real time PCR protocol utilizing the easyMAG and the LightCycler 480/II, both very common devices in viral molecular diagnostics. For clinical validation, 123 CSF specimens and 40 reference samples from national interlaboratory comparisons were examined with both methods, resulting in 97.6% and 100% concordance for CSF and reference samples, respectively. Utilizing the BD Max platform revealed sensitivities of 173 (CI 95%, 88-258) copies/ml for HSV1, 171 (CI 95%, 148-194) copies/ml for HSV2 and 84 (CI 95%, 5-163) copies/ml for VZV. Cross reactivity could be excluded by checking 25 common viral, bacterial and fungal human pathogens. Workflow analyses displayed shorter test duration as well as remarkable fewer and easier preparation steps with the potential to reduce error rates occurring when manually assessing patient samples. This protocol allows for a fully automated PCR assay on the BD Max platform for the simultaneously detection of herpesviridae from CSF specimens. Singular or multiple infections due to HSV1, HSV2 and VZV can reliably be differentiated with good sensitivities. Control parameters are included within the assay, thereby rendering its suitability for current quality management requirements.

No MeSH data available.


Related in: MedlinePlus