Many of the current reverse transcription (RT)-PCR assays for the detection of hepatitis C virus (HCV) RNA are multistep processes which use multiple enzymes and buffers. The assays are also often suboptimal, requiring nested amplification to achieve the desired levels of sensitivity. As a result, these assays are cumbersome and prone to false-positive results. The susceptibility to contamination is further aggravated by the lack of carryover controls.
Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, Netherlands.The polymerase chain reaction (PCR) is used to detect hepatitis C virus (HCV) RNA, and the results of this assay may have a bearing on management of patients. We tested 31 laboratories for performance of HCV PCR with a coded panel that comprised 4 HCV-positive plasma samples, 6 HCV-negative samples, and two dilution series of HCV-positive plasma. 15 (48%) laboratories had faultless results with both dilution series, and 16 (52%) laboratories reported erroneous results with one or both series. 10 (32%) laboratories had faultless results when testing undiluted plasma samples, 11 (35%) produced a false-negative result with a weak-positive sample, and 10 (32%) produced false negative and/or false positive results. Only 5 (16%) laboratories performed faultlessly with the entire panel of samples. Reports of presence of HCV should be interpreted with care until reliable HCV-RNA detection becomes widely available.
http://www.findarticles.com/cf_dls/m0BPG/7_15/76609752/p1/article.jhtmlTo confirm ongoing viremia, testing for HCV RNA by polymerase chain reaction (PCR) is recommended. Because testing for HCV RNA by PCR is expensive, is difficult to perform, and can yield false-positive and false-negative results depending on the laboratory used, it is not recommended as a first-line test for the diagnosis of hepatitis C in most patients.
RT-PCR is a very powerful tool for detecting relatively low amounts of genetic material (RNA or DNA). The basis of this technique is the amplification of a target piece of nucleic acid several million times so that this target becomes measurable. Due to the extreme sensitivity of this technique, however, the slightest contamination can lead to a false positive result.
Clearly, the combination of reverse-transcriptase polymerase chain reaction (RT-PCR) and PCR amplification of viral complementary DNA (cDNA) has had a pivotal role in the identification of the virus responsible for the majority of non-A non-B hepatitis cases. However, although it has allowed the molecular characterization of this virus, the unique sensitivity of RT-PCR has also created a number of problems. For example, a cell culture system for the propagation of hepatitis C virus (HCV) would be of great benefit. However, formal proof of replication in such systems has not been easy to obtain. The use of "strand-specific" RT-PCR to detect the replicative intermediates (minus-strand RNA) of HCV has been taken as evidence for viral replication. However, recent developments have shown that strand-specific RT-PCR is fraught with problems, and many early studies claiming low-level viral replication based on the detection of minus-strand RNA may be flawed. Potential causes of nonspecific amplification of positive-strand RNA sequence by using tagged RT-PCR. (A) Conventional reverse transcription of RNA into cDNA at 42°C with the minus-strand specific tagged HCV primer can lead to nonspecific synthesis of cDNA from positive-strand RNA by virtue of (a) self-priming, (b) random prining, or (c) false priming. (B) These nonspecific cDNAs along with some of the tagged primer will be transferred into the subsequent PCR reaction mix. The tagged HCV primer (through its HCV-complementary sequence) can now use the nonspecifically amplified cDNA as template during the first cycle of the PCR reaction. (C) In further PCR cycles, amplification of the target by the PCR primers (tag primer, which lacks HCV sequence, and the downstream HCV primer) leads to a false-positive result.
"Kary Mullis, inventor of PCR, won a 1993 Nobel prize for his billion-dollar invention, which has become indispensable to any genetics lab. It is ironic that one of the first applications of PCR was to detect HIV, considering that Mullis himself doesn’t believe his invention is capable of this. Mullis states the problem is PCR is too efficient – it will amplify whatever DNA is in the sample, regardless of whether that DNA belongs to HIV or a contaminant. And how do you decide which part of the amplified material could be HIV and which part the contaminant(s), if you couldn’t detect HIV in the sample without using PCR?"