Hepatitis C Virus (HCV) PCR Test Procedure (Artus HCV RG PCR Kit)

Principle:

The pathogen detection by PCR is based on the amplification of specific regions of the pathogen genome. In real time PCR, the amplified product is detected via the fluorescent dyes. These are usually linked to the oligonucleotide probes which bind specifically to the amplified product.

RNA Extraction Procedure: (See DNA Extraction using QIAmp DSP Virus Kit DNA/RNA Extraction Procedure)

Master Mix Preparation: 

• Please use the following pipetting scheme:

	number of samples	1
	HCV-RG MasterA	12 ul
Prep. of	HCV-RG MasterB	18 ul
Mastermix	HCV-RG IC	2 ul
	Total volume	32 ul
Prep of	Mastermix	30 ul
PCR assay	Sample	20 ul
	Total volume	50 ul

• Mix the solution by pipetting up and down. Close the PCR tubes and ready to load the tubes in Rotorgene 6000 for amplification. 

Interpretation of Results:

• A signal is detected is fluorescence channel Cycling Green or Cycling A.FAM., 

         The result is positive: the sample contains HCV-RNA. 

• In fluorescence channel Cycling Green or Cycling A.FAM no signal; is detected. At the same time, a signal from the Internal control appears in the Cycling Orange channel or the Cycling A.ROX channel.

         No HCV-RNA detected. It can be considered negative. 

• No signal is detected in the Cycling Green or Cycling A.FAM channels, nor in the Cycling Orange or the Cycling A.ROX channels. 

         No result can be concluded. Presence of PCR inhibitions are suspected, for repeat extraction.

Reference Values:

• HCV- RNA >34 IU/ml: The result is within the determined test range. 

                                              The detection probability of HCV- RNA is >95%.

                                              The positive test result is statistically ensured.

• HCV- RNA <34 IU/ml: The result is outside the determined test range. 

                                              The reproducibility of the positive result is not assured.

• HCV-RNA negative. : No HCV- RNA was detected.