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In-house designed Multiplex Real-Time PCR Assay Developed for Epstein - Barr virus and Cytomegalovirus DNA detection
Ghinwa S Majid1, Ahmed S Abdulamir2, Iman M Aufi3
1Biology, Alnahrain University, College of sciences, Baghdad, 10001, Iraq.
Background:
The problem of transmissible human herpesvirus infections through blood donation has not been completely resolved by serological testing due to issues such as delayed in seroconversion or false-negative results. This study aimed to develop a cost-effective, In-house designed multiplex real-time PCR assay for the simultaneous detection of Epstein-Barr virus (EBV) and cytomegalovirus (CMV) DNA in pooled blood donor samples.
Methods:
A total of 1000 whole-blood samples were collected from apparently healthy Iraqi blood donors and pooled into mini pools 10(MP10) i.e. 100 MP10 tested by a commercial CMV and EBV multiplex- qPCR detection kit; then retested by In-house designed multiplex qPCR. Fifteen patients who were positive for CMV and fifteen for EBV were considered as positive groups and fifteen healthy individuals as negative groups used to estimate the validity and feasibility of the In-house designed kit.
Results:
Blood donors who tested by the commercial kit were 42% MP10 positive for EBV and 4% positive for CMV while the In-house designed multiplex qPCR results were 40% and 6% for EBV and CMV, respectively. The specificity and sensitivity of the In-house designed kit for the detection of CMV were 97.92% (95% CI 92.6 to 99.7), 100% (95% CI 39.7 to 100), for EBV were 100% (95% CI 93.84 to100.00) and 95.24 % (95% CI 83.84 to 99.42), respectively.
Conclusion:
The rate of EBV was high within Iraqi blood donors while the CMV was moderately low and the In-house designed multiplex qPCR was sensitive and specific for CMV and EBV DNA detection at a much lower cost than that of the commercial, about 10% of the commercial cost. Such a cost-effective and sensitive test is crucial in reducing blood transfusion-associated diseases.
