Development of real-time PCR array for simultaneous detection of eight human blood-borne viral pathogens.
BACKGROUND
Real-time PCR array for rapid detection of multiple viral pathogens should be highly useful in cases where the sample volume and the time of testing are limited, i.e. in the eligibility testing of tissue and organ donors.


RESULTS
We developed a real-time PCR array capable of simultaneously detecting eight human viral pathogens: human immunodeficiency virus types 1 and 2 (HIV-1 and -2), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell leukemia virus-1 and -2 (HTLV-1 and -2), vaccinia virus (VACV) and West Nile virus (WNV). One hundred twenty (120) primers were designed using a combination of bioinformatics approaches, and, after experimental testing, 24 primer sets targeting eight viral pathogens were selected to set up the array with SYBR Green chemistry. The specificity and sensitivity of the virus-specific primer sets selected for the array were evaluated using analytical panels with known amounts of viruses spiked into human plasma. The array detected: 10 genome equivalents (geq)/ml of HIV-2 and HCV, 50 geq of HIV-1 (subtype B), HBV (genotype A) and WNV. It detected 100-1,000 geq/ml of plasma of HIV-1 subtypes (A - G), group N and CRF (AE and AG) isolates. Further evaluation with a panel consisting of 28 HIV-1 and HIV-2 clinical isolates revealed no cross-reactivity of HIV-1 or HIV-2 specific primers with another type of HIV. All 28 viral isolates were identified with specific primer sets targeting the most conserved genome areas. The PCR array correctly identified viral infections in a panel of 17 previously quantified clinical plasma samples positive for HIV-1, HCV or HBV at as low as several geq per PCR reaction.


CONCLUSIONS
The viral array described here demonstrated adequate performance in the testing of donors' clinical samples. Further improvement in its sensitivity for the broad spectrum of HIV-1 subtypes is under development.
Tissue Microbiology Laboratory, Division of Cellular and Gene Therapies, Office of Cellular, Tissue and Gene Therapy, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, United States of America.
Natalia
N
Pripuzova
Richard
R
Wang
Shien
S
Tsai
Bingjie
B
Li
Guo-Chiuan
GC
Hung
Roger G
RG
Ptak
Shyh-Ching
SC
Lo
D052160:Benzothiazoles; D017848:Blood-Borne Pathogens / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D017977:Deltaretrovirus / Q000235:genetics*; D003959:Diamines; D006678:HIV / Q000235:genetics* / Q000302:isolation & purification; D006518:Hepatitis Viruses / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D009930:Organic Chemicals; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014616:Vaccinia virus / Q000235:genetics* / Q000302:isolation & purification; D014777:Virus Diseases / Q000175:diagnosis*; D014902:West Nile virus / Q000235:genetics* / Q000302:isolation & purification
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I