Development of an improved product enhanced reverse transcriptase assay.
A PCR based reverse transcriptase (RT) assay was developed that has 10(4)-fold higher sensitivity than conventional nucleotide incorporation assays and allows discrimination between false positive results generated by cellular polymerases and positives resulting from authentic RT activity. Recently, several reverse transcriptase (RT) assays have been developed where a reverse transcriptase reaction is performed on an RNA template/DNA primer combination. A specific region of the cDNA product is then amplified by the polymerase chain reaction to increase the sensitivity of cDNA detection. These reverse transcriptase assays up to 10(6)-fold more sensitive at detecting retroviruses than conventional methods. The drawback to these assays with increased sensitivity is the increased incidence of false positive results generated by cellular polymerases that can reverse transcribe. The MS2 bacteriophage RNA template and primers from one of the recently developed assays were used as the basis to develop the assay. A simple high resolution agarose gel was used as the endpoint for the assay without compromising sensitivity. In addition, the pH of the RT reaction was lowered to pH 5.5, the RT incubation was 1 h, and protease inhibitors were added to the RT reaction components. These modifications yield an assay that can discriminate between authentic RT activity and contaminating cellular polymerases.
Chang
A
A
Microbiological Associates, Rockville, MD 20850, USA. achang@microbio.com
Ostrove
J M
JM
Bird
R E
RE
D016475:3T3 Cells; D000818:Animals; D002474:Cell-Free System / Q000737:chemistry; D002478:Cells, Cultured / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D005189:False Positive Reactions; D006863:Hydrogen-Ion Concentration; D017909:Levivirus / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011925:Rauscher Virus / Q000737:chemistry / Q000201:enzymology; D012192:Retroviridae Infections; D012680:Sensitivity and Specificity; D013698:Templates, Genetic
D017931:DNA Primers; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase