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MycAssay™ Pneumocystis
What is the kit used for?
MycAssayTM Pneumocystis is indicated for use by qualified laboratory professionals for the qualitative detection of Pneumocystis jirovecii genomic DNA extracted from respiratory specimens from the human lower respiratory tract (e.g. bronchial samples) as an aid to diagnosis in adult patients suspected of having P. jirovecii pneumonia.
What is the Pneumocystis molecular target in the assay?
A portion of the mitochondrial ribosomal large sub-unit (rLSU)
You recommend the use of MycXtra® for fungal DNA extraction. Can DNA extracted with other kits be used successfully with the MycAssay TM Pneumocystis Real-Time PCR kit?
Studies using the MycAssay TM Pneumocystis Real-Time PCR kit with clinical samples enable us to define a clinical cut off, and also sensitivity and specificity of the assay in a clinical setting. We have done this using MycXtra® so the sensitivity and specificity data we quote are only valid for the specific situation where you use clinical respiratory samples, extract Pneumocystis DNA with MycXtra® and analyse with MycAssayTM Pneumocystis on the Cepheid SmartCycler.
However, MycAssayTM Pneumocystis works perfectly well with solutions of DNA whether obtained by MycXtra® or any other extraction system. The analytical characteristics of the kit remain the same but, because we do not know how well these other systems extract Pneumocystis DNA, remove inhibitors or are free from Pneumocystis contamination, we cannot give clinical specificity and sensitivity for these DNA extraction systems. In these cases the final result which states ‘positive’ or ‘negative’ should be ignored; the Ct values on the Pneumocystis channel indicate the sample result. The Internal Amplification Control (IAC) and the kit Positive and Negative controls are still valid.
Can the MycAssay TM Pneumocystis kit be used on any other Real-Time platforms apart from the Cepheid SmartCycler?
Yes, it has been valdiated for use on the AB7500, the Roche LightCycler® 2.0 and the Stratagene Mx3000 series. If you would like to use the kit as an RUO product, please click here for more information.
Can MycAssayTM kits be run on the Roche LightCycler 1.5?
The MycAssayTM PCR kits have been designed, optimised and validated to work on the market leading real-time PCR platforms (Cepheid SmartCycler, Roche LightCycler 2.0, ABI 7500 and Stratagene MX3000 series instruments).Some older PCR machines are not compatible with MycAssay™ products, such as the Roche LightCycler 1.5.
The MycAssayTM kits will not work on the Roche LightCycler 1.5, because the LightCycler 1.5 is unable to specifically detect the HEX dye used on the Internal Amplification Control (IAC) beacon. The LightCycler 1.5 has three channels for detecting fluorescence: 530nm, 645nm, & 710nm. In contrast, the LightCycler 2.0 has six channels 530nm, 555nm, 610nm, 640nm, 670nm & 710nm. The MycAssayTM kits require the use of the 530nm channel to detect FAM dye for the target and the 555nm (referred to as 560nm in the IFU) channel to detect the HEX dye for the Internal Amplification Control (IAC). Therefore, due to limitations in the number of channels the MycAssayTM kit cannot be applied to the LightCycler 1.5 but can run successfully on the LightCycler 2.0.
How long does the assay take to perform?
Less than 4 hours from sample receipt to result:
| Fungal DNA extraction from clinical sample | ~2 hours |
| Real-Time PCR set-up | 15 minutes |
| Real-Time PCR run | 1.5 hours |
| Results analysis | 15 minutes |
What are the analytical characteristics of the assay?
Limit of Blank (LoB) = Ct 39.0
The LoB was determined analytically using samples known not to contain Pneumocystis DNA. Based on these data, anything with a Ct ≤38.9 is positive for the presence of Pneumocystis DNA.
Limit of Detection (LoD) = <35 copies of the mitochondrial rLSU.
This value was determined using a recombinant DNA plasmid harbouring the target sequence. The Pneumocystis target sequence is mitochondrial, therefore there will be numerous copies per cell, but it is not known how many.
What are the sensitivity and specificity values?
MycAssayTM v Immunofluorescent (IF) microscopy
|
|
IF Microscopy Positive |
IF Microscopy Negative |
|
|---|---|---|---|
|
MycAssayTM Positive |
45 |
8 |
PPV 0.85 |
|
MycAssayTM Negative |
2 |
33 |
NPV 0.94 |
|
|
Sensitivity 0.96
|
Specificity 0.80
|
|
Are there any substances which may inhibit the assay PCR?
We tested 17 different compounds at clinically relevant concentrations, the majority of which were antifungal drugs which may be present in this patient population. None of the 17 substances we tested inhibited the assay. A full list of these substances tested can be found in the kit Instructions for Use.
For any further questions, please contact [email protected]