Design and use of signature primers to detect carry-over of amplified material.
Signature primer pairs designed for use with the polymerase chain reaction have been developed which can determine if a positive result originated from the intended target nucleic acid or from so-called "carry-over" contamination of previously amplified DNA. The 3' ends of each signature primer, SK339/341, SSK110/111, and SSK58/59 contain a viral specific sequence complementary to regions of either HIV-1, HTLV-I and II respectively. The 5' ends of each primer contain a non-human, non-viral (NHNV) signature sequence including restriction endonuclease sites for subsequent cloning. A fourth set of primers, SK338/340, consist solely of these NHNV sequences and are designed to anneal to any product previously amplified by the viral-specific signature primers. These primers were tested against their corresponding positive and negative DNA targets, to determine their specificity and sensitivity. As expected, the viral-specific signature primers detected the retroviral infected samples while no detectable amplification occurred in negative DNA controls. Primers SK338/340 did not amplify any viral positive or negative template DNA's. Samples spiked with amplified material generated from the viral-specific signature primers could be specifically amplified by the NHNV primers SK338/340. Primers SK338/340 were determined to be more sensitive than the viral-specific signature primers, ensuring the detection of extremely low amounts of carryover. This strategy may be useful in developing other retroviral or non-retroviral primers with a built-in signature sequence that can differentiate false positives from true positives in a subsequent confirmatory test.
Abbott
L Z
LZ
Department of Medicine, SUNY Health Science Center, Syracuse 13210.
Spicer
T
T
Bryz-Gornia
V
V
Kwok
S
S
Sninsky
J
J
Poiesz
B
B
D016477:Artifacts; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004279:DNA, Viral / Q000302:isolation & purification*; D004866:Equipment Contamination; D005189:False Positive Reactions; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D015368:Human T-lymphotropic virus 1 / Q000235:genetics / Q000302:isolation & purification*; D015367:Human T-lymphotropic virus 2 / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004279:DNA, Viral