POLYMERASE CHAIN-REACTION AND TRANSFUSION MICROBIOLOGY

Polymerase chain reaction (PCR) involves alternate denaturing and re-annealing of DNA in test samples in the presence of appropriate oligonucleotide primers complementary to opposite strands of the target DNA together with a heat-stable DNA polymerase, Mg2+ and the four nucleotide triphosphates. DNA target segments can be 'amplified' ten-millionfold by 25-35 such cycles. Even greater amplification (approximately 10(12)-fold) with enhanced specificity can be obtained by a second set of amplification cycles using a further pair of 'nested' primers sited within the DNA sequence defined by the original primers. PCR can be applied to the study of the whole range of transfusion-transmitted infections, both plasma and cell associated; RNA viruses can be analyzed if a DNA copy is made from the viral RNA by treatment with reverse transcriptase. In a transfusion context, the retroviruses (HIV-1, HIV-2, HTLV-I, HTLV-II), HCV and HBV have been the viruses most intensively subjected to PCR analysis. The advantages of PCR in this context include its ability to detect virus during the 'window period' or seronegative stages of infections and its value as a marker for viraemia and for the detection of viruses in products made from large pools of plasma. True immunity may also be differentiated from persistent infection in the presence of antibody. Similarly, PCR can overcome problems of diagnosis of acute infection caused by the presence of passively transferred antibody. Detailed strain differentiation is also possible by PCR, in conjunction with sequencing or with the aid of restriction endonucleases. However, PCR has its limitiations: The detection of nucleic acid does not necessarily equate with infectivity; (1) the timing of sample collection maybe critical for detecting transient viraemia; (2) sample storage (especially with RNA viruses) must be optimal for nucleic acid integrity; (3) highly conserved primers must be chosen for detection of the widest range of strain variants and (4) the small volume of test sample does not compare with the volume of inoculum represented by transfused blood components. Furthermore, PCR has operational drawbacks. False positivity due to endogenous human DNA sequences may occur if primers are not carefully chosen, and cross-contamination is an ever present threat. PCR is also labour intensive and relatively expensive. Nevertheless, there is considerable potential for the simplification of DNA extraction procedures and for the streamlining of amplification and DNA visualisation methods. In a pre-transfusion screening context however, the potential problems of carry-over and cross-contamination remain formidable, and at present the greatest value of PCR lies in validation and research fields in which it promises to have an exciting and productive future.