Serological tests
A virus is an intracellular germ which is characterized by its inability to reproduce by division, and therefore requires a host cell for its multiplication. It is composed of a nucleic acid molecule (either DNA or RNA, single or double stranded, linear or circular, mono or polysegmented) surrounded by a protein shell called the capsid, and sometimes a envelope. Viral Genome Screening (VGD) involves looking for the nucleic acid of viruses.
This technique makes it possible to detect very recent infections, even before the antibodies are detectable by serological tests. It has thus made it possible to reduce the silent window or serological window (period in which the germ of an infected person is undetectable), by 11 days for HIV-1 from 22 to 11 days on average, by 49 days for HCV from 58 to 9 days on average, and from 25 days for HBV from 50 days to 25 days.
DGV technique
The realization of the Viral Genome Screening is based on three steps:
Sample preparation: nucleic acid extraction
Amplification of a specific sequence of the genome of the virus sought by PCR (polymerase chain reaction) or TMA (Transcription Mediated Amplification)
Detection of amplified products
Sample preparation
The preparation of samples from blood donors consists of the extraction of nucleic acids by the lysis of cell membranes and viral envelopes by an enzymatic (proteinase K) or chemical (guanidium thiocyanate) route. The nucleic acids are then separated from the reaction medium and plasma residues using silica beads or by capture using probes (short DNA or RNA fragments), the sequence of which is complementary to a specific sequence of the nucleic acid to be extracted.
PCR amplification
PCR amplification is a real exponential multiplication of a precise nucleotide sequence by a sequence of cycles:
Thermal denaturation
Hybridization with a pair of primers
Elongation of each primer by Taq polymerase
Amplification by TMA
This amplification technique makes it possible, from a double-stranded DNA, to obtain billions of copies of RNA at a constant temperature. This multiplication is carried out by the use of T7 RNA polymerase. The difference, compared to PCR amplification, is the possibility of simultaneously detecting the genomes of the HIV1 and HCV viruses in the same reaction.
Nucleic acid detection
Before the detection of nucleic acids, pools (mixture of blood from several donors) are made to save time in the analyzes and allow the transfusion of blood products, except for DOM. For the analyzes carried out by TMA, the pools are composed of 8 samples whereas for the PCR, 24 samples are analyzed per pool.
For the PCR technique, the detection of nucleic acids is carried out by probes (short DNA or RNA fragments) specific for viral RNA, attached to magnetic particles. Biotin-labeled amplicons are added, followed by an avidin-peroxidase conjugate. After washing, a substrate is added in order to read the results.
For the TMA technique, amplicons are detected using probes labeled with acridinium ester. The reading is carried out by chemiluminescence in an alkaline medium.
DGV limits
DGV made it possible to detect the presence of virus, before the appearance of antibodies in the donor. But when the number of viruses is too low, DGV cannot detect the presence of these viruses. This may be the case at the start of contamination or during seroconversion.
The search for the nucleic acid of the virus is dependent on the virus (HIV-2 not detected) and on the genetic variations of the viral strains. Some genetic variations of the virus cannot be detected by DGV.
Finally, the nucleic acid may have been degraded during sample collection, due to the presence of inhibitors in the sample, such as heparin.