Hepatitis C Virus Genotype, Serum
Method Description Describes how the test is performed and provides a method-specific reference
The Abbott mSample Preparation System kit is used with the Abbott m2000sp, an automated sample preparation system using the magnetic microparticle processes, is used to extract and purify viral nucleic acids from human serum specimens. An internal control is incorporated in the nucleic acid extraction and purification procedure for processing the assay controls and clinical specimens. After capture of nucleic acids onto magnetic microparticles, the microparticles are washed to remove unbound sample components. Then, the bound nucleic acids are eluted from the microparticles and the eluates are transferred to a 96-well microtiter plate containing PCR mastermix reagents for amplification and detection.
Amplification, Detection, and Genotyping:
The Abbott RealTime HCV Genotype II RUO assay uses 4 sets of PCR primers. One set of primers targets a sequence within the 5' untranslated region (UTR) of the hepatitis C virus (HCV) genome. This primer set is designed to amplify all HCV isolates. The second primer set is designed to amplify the non-structural (NS) 5b region of genotype 1a. The third HCV primer set is designed to amplify the NS5b region of genotype 1b. An internal control primer set is included to amplify a portion of the hydroxypyruvate reductase gene of the pumpkin plant, Cucurbita pepo. The assay positive control is an Armored RNA particle diluted in negative human plasma.
During the amplification reaction, cDNA is generated from the target RNA sequence by the reverse transcriptase activity of the thermostable rTth DNA polymerase. First, the HCV and internal control reverse primers anneal to their respective target sequences and are extended during a prolonged incubation period. After a denaturation step, in which the temperature of the reaction is raised above the melting point of the double-stranded cDNA:RNA product, a second primer anneals to the cDNA strand and is extended by the DNA polymerase activity of the rTth enzyme to create a double-stranded DNA product. During each round of thermal cycling, amplification products dissociate to single strands at a high temperature, allowing primer annealing and extension as the temperature is lowered. Exponential amplification of the product is achieved through repeated cycling between high and low temperatures, resulting in a billion-fold or greater amplification of target sequences. Fluorescent probes specific for HCV genotypes 1 to 6 and subtypes 1a and 1b anneal to the amplified sequence products in 3 separate reaction wells for each specimen. Composite results generated from these 3 reaction wells determine the HCV genotype present in a given clinical specimens.(Package insert: Abbott RealTime HCV Genotype II RUO Test, Abbott Molecular Inc., Des Plaines, IL, 8/2010)
Day(s) and Time(s) Test Performed Outlines the days and times the test is performed. This field reflects the day and time the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time required before the test is performed. Some tests are listed as continuously performed, which means assays are performed several times during the day.
Monday through Friday; 8 a.m.
Analytic Time Defines the amount of time it takes the laboratory to setup and perform the test. This is defined in number of days. The shortest interval of time expressed is "same day/1 day," which means the results may be available the same day that the sample is received in the testing laboratory. One day means results are available 1 day after the sample is received in the laboratory.
Maximum Laboratory Time Defines the maximum time from specimen receipt at Mayo Medical Laboratories until the release of the test result
Specimen Retention Time Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded
Performing Laboratory Location The location of the laboratory that performs the test