Application of RT-Bst to enhance detection of pathogenic viruses of the respiratory tract.
Inefficiency of RT-PCR can be associated with the suboptimal process of reverse transcription as only 40-80% of RNA is converted to cDNA. We employed a novel method, RT-Bst, to enrich the concentration of cDNA for subsequent multiplex PCR detection of selected RNA viruses. The RT-Bst method amplifies cDNA through reverse transcription of viral RNA using reverse transcriptase and amplification of cDNA using Bst DNA polymerase. Viral RNA was extracted from 25 nasopharyngeal samples for detection of influenza A, B and C; parainfluenza 1-4; human coronaviruses 229E and OC43; respiratory syncytial virus (RSV) and rhinovirus. Both multiplex one-step RT-PCR and RT-Bst PCR were used to compare their performances for detection of virus sequences. These findings were compared with routine laboratory detection. When using RT-Bst PCR, 28% of samples yielded a viral pathogen compared to 20% with RT-PCR and 12% using routine diagnostic tests. RT-Bst PCR was shown to have particular utility in the detection of RSV RNA as this was present in 20% of the samples studied compared to 8% when using RT-PCR. For one patient, RT-Bst PCR was able to detect RSV five days earlier than conventional hospital diagnostic testing. RT-Bst and RT-Bst PCR can be used as alternative approaches to reverse transcription and one-step RT-PCR, respectively, for sequence-independent amplification of RNA virus sequences and a larger scale analysis of this new diagnostic approach is warranted.
M S
MS
Kabir
M O
MO
Clements
M
M
Atkins
P T
PT
Kimmitt
D001426:Bacterial Proteins / Q000737:chemistry; D017934:Coronavirus / Q000235:genetics* / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D009975:Orthomyxoviridae / Q000235:genetics* / Q000302:isolation & purification; D012136:Respiratory Syncytial Viruses / Q000235:genetics* / Q000302:isolation & purification; D012137:Respiratory System / Q000473:pathology / Q000821:virology; D012141:Respiratory Tract Infections / Q000175:diagnosis* / Q000473:pathology / Q000821:virology; D018108:Respirovirus / Q000235:genetics* / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D012229:Rhinovirus / Q000235:genetics* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D014777:Virus Diseases / Q000175:diagnosis* / Q000473:pathology / Q000821:virology
D001426:Bacterial Proteins; D004259:DNA-Directed DNA Polymerase