DETECT cCMV IN A SINGLE DRIED BLOOD SPOT PUNCH

NeoMDx^™ cCMV Real-time PCR Assay

The research use only NeoMDx^™ cCMV real-time PCR assay is a multiplexed, real-time qPCR test delivering qualitative detection of congenital cytomegalovirus (cCMV) and the RPP30 (Ribonuclease P/MRP Subunit P30, RNase P) gene in DNA from dried blood spots (DBS).

Conserves valuable samples by only using one blood spot for analysis
Leverages universal DBS workflows
Residual DNA isolated for other analyses can be used in this assay

The NeoMDx^™ cCMV real-time PCR assay detects this cCMV from a single 3.2 mm DBS punch. Since DBS samples are available but limited, the ability to analyze a single DBS punch for cCMV offers advantages over other assays which either use saliva as the sample type (which is difficult to obtain) or require many DBS punches to detect the virus.

For research use only. Not for use in diagnostic procedures.

cCMV REAL-TIME PCR WORKFLOW

The NeoMDx^™ cCMV real-time PCR assay leverages a standard real-time qPCR workflow starting with dried blood spots. Additionally, residual DNA isolated from other PCR assays can be used in this workflow, further simplifying the detection of cCMV and conserving existing DBS real estate for future analysis.

cCMV Real-time qPCR Primers, Probes, & Controls

The NeoMDx^™ cCMV real-time PCR assay uses target sequence-specific primers and TaqMan^™ probes (Thermo Fisher Scientific) to amplify and detect cCMV DNA and RPP30, a human gene used as an internal control. The qPCR sample input is extracted DNA from a single dried blood spot (DBS) punch using NeoMDx^™ Elution Solution.

The oligonucleotide primers and probes for detection of cCMV are selected from the UL122 gene region (regulatory protein IE2).

The assay also uses a dUTP/UNG carryover prevention system to avoid contamination by PCR products and erroneous results.

There are three DBS kit controls: C1 (CMV negative control), C2 (CMV low positive control), and C3 (CMV high positive control). All three controls are punched and extracted along with the sample before running on the real-time qPCR instrument to ensure the entire workflow is working as intended.

Real-time qPCR Instrument Compatibility

The probe for detection of cCMV is labeled with FAM. The probe for detection of RPP30 is labeled with Cy5. Any Real-time qPCR Instruments with FAM^™ and Cy5 channels can be used with this assay. These include, but are not limited to:

Eonis Q (2044-0020 for 96, and 2045-0020 for 384)
Applied Biosystems^™ QuantStudio^™ 3 Real-Time PCR System
Applied Biosystems^™ QuantStudio^™ 5 Real-Time PCR System
Applied Biosystems^™ QuantStudio^™ 6 Real-Time PCR System
Applied Biosystems^™ QuantStudio^™ 7 Real-Time PCR System
Applied Biosystems^™ QuantStudio^™ Dx Real-Time PCR System
Analytik Jena^® Tower3/qTower3 3 G 96 Real-Time PCR System
Analytik Jena^® qTower3 84/qTower3 84 G Real-Time PCR System
Applied Biosystems^™ 7500 Real-Time PCR System
Applied Biosystems^™ 7500 Fast Dx Real-Time PCR System
Bio-Rad^® CFX384^™ Real-Time PCR Detection System
Bio-Rad^® CFX96^™ Touch Real-Time PCR Detection System
Roche^® LightCycler^® 480 system

Straight-forward Result Interpretation:

	Ct
Control/Sample Name	CMV (FAM)	RPP30 (Cy5)
Negative control (NTC)	Undet or > 40	> 35
C1	Undet or > 40	[20, 24]
C2	[32, 36]	[20, 24]
C3	[27, 31]	[20, 24]
Sample	≤ 40	≤ 35

Undet: Undetermined.
The specification is defined based on QuantStudio^TM 7 Flex with the analysis settings as: baseline (3-15), FAM threshold = 0.4, Cy5 threshold = 0.2. It is subjected to change for different real-time PCR instruments (supplier, brand, etc.)

FAM (CMV)	Cy5 (RPP30)	Results
N	N	Invalid. Sample needs to be re-tested by repeat PCR if there is sufficient extracted DNA. Otherwise, sample needs to be re-tested by re-extraction or re-collection from subject.
Y	Y/N	CMV detected.
N	Y	CMV not detected.