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The diagnostics of the hepatitis C virus with usage of methods of the molecular biology
JAKUBCOVÁ, Markéta
Hepatitis C is a virus caused liver irritation, which leads to hepatocelular carcinoma in more than a quarter of cases. Six different genotypes with at least fifty different subtypes are known (3). Hepatitis C virus rapidly undergoes mutation, which causes every infected person to have more of slightly different variants of the virus in their body. Consequently, the immune system often loses control over the virus, which can lead to the observed chronicity. Qualitative confirmation is important for determination of the presence of the virus. Quantitative confirmation, on the other hand, is crucial for commencing and monitoring of the treatment. (20) The most often examined species is blood serum, or plasma. HCV can be diagnosed directly, or indirectly. Confirmation of the presence of anti-HCV antibodies (indirectly) by Elisa method is routine and very basic screening in most laboratories. Detectable anti-HCV antibodies occur three weeks to six months after the primoinfection (every person?s immune system reacts differently). This indirect test is usually negative in early phases of infection. (21) For direct confirmation of the virus, solely PCR ? polymerase chain reaction ? methods are used. In the first phase, it is necessary to isolate RNA of the virus from the biological material. This can be done either by a special isolation instrument, or manually, using special isolating kits. Use of different methods may result in different yield and purity of RNA. In my work, I used only hand column isolation. After obtaining the RNA of the virus, qualitative confirmation follows. In my case, an ?In house? method was used ?the RT PCR (reverse transcription PCR) followed by the PCR amplification of specific segment of viral RNA, which are visualized by gel electrophoresis in presence of ethidium bromide, which works as an intercalation species. Double step PCR is used. In the first step, reverse transcription progresses for 30 minutes at 50°C. In the presence of RNase inhibitor and reverse transcriptase enzyme RNA is overwritten to cDNA, which is subsequently multiplicated using specific primers. This process is a part of one PCR reaction, which takes place in the Thermocycler in the presence of specific primers, water, buffer, nucleotides, and specific enzyme. For the second round of Nested PCR two different sequences of primers are used. PCR is used for enhancing the sensitivity and specifity of the method. Products of the PCR are then visualized on the agarose gel with intercalation agent (most commonly ethidium bromide).(3) Quantitative determination of viremia level is carried out with Real Time PCR. Detection proceeds in real time using fluorescence dyes. Dyes are usually bonded onto oligonucleotide probe, which specifically bonds to a PCR amplificate. Measuring the intensity of fluorescence enables confirmation and quantification of the products. The system is also able to identify potencial PCR inhibition with internal control. After addition of standards of known concentration, we can work out a calibration curve and use it to quantify measured samples (19). In this work, two isolation kits are compared: a) High Pure Viral RNA Kit (Roche) ? kit used for isolation viral RNA from biological material (serum, plasma). In the first phase, biological material is decomposed, RNA is collected on the column with membrane, washed multiple times and eluted to water for use in Real Time PCR (8) b) QIAamp Viral RNA mini Kit (Qiagen) ? kit used for isolation of viral RNA from blood plasma, or serum. Firstly, biological material is decomposed, RNA is collected on the column with membrane, washed multiple times and eluted to water for use in Real Time PCR (13).The samples isolated with the QIAamp Viral RNA Kit (Qiagen) have shown higher values of measured viral RNA after quantification.
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