sybr-green-i and Orthomyxoviridae-Infections

Topics: Animals; Benzothiazoles; Diamines; Humans; Influenza A virus; Influenza A Virus, H1N1 Subtype; Influenza, Human; Orthomyxoviridae Infections; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Swine

The novel influenza A(H1N1) virus that emerged recently in Mexico has spread rapidly to many countries and initiated a human pandemic. It would be interesting to determine whether the virus has existed in, or will spread to, the swine population. However, it is difficult to differentiate the virus from some swine influenza viruses. In this study, a SYBR Green I real-time RT-PCR assay was designed for detection and differentiation of influenza A(H1N1) virus from some swine influenza viruses, by comparing the amplification of two pairs of primers corresponding to influenza A(H1N1) virus and some swine influenza viruses, respectively. The assay was evaluated using online analysis, identified influenza viruses and clinical samples. The results indicated that the assay has high sensitivity and specificity to detect influenza A(H1N1) virus, and is able to differentiate it from some swine influenza viruses. This, in turn, could provide essential epidemiological information for risk analysis and decision making in combating the disease, and stimulate research to differentiate pathogens similar to each other using the same method.

Topics: Animals; Benzothiazoles; Diamines; DNA Primers; Influenza A Virus, H1N1 Subtype; Organic Chemicals; Orthomyxoviridae Infections; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Swine; Swine Diseases

Routine laboratory diagnosis of infectious salmon anaemia virus (ISAV) infection is primarily by reverse transcription polymerase chain reaction (RT-PCR) because of the high sensitivity and rapid turnaround time of the test. This paper describes methods for highly reproducible absolute viral load measurements using external standard curves generated with either ISAV recombinant plasmid DNA (pDNA) standards or transcribed RNA standards prepared by in vitro transcription with T7 RNA polymerase, and using a two tube real-time or quantitative (q)RT-PCR with SYBR Green I chemistry and a single tube qRT-PCR with TaqMan probe chemistry. When applied to virus samples of known virus titer for the highly pathogenic ISAV strain NBISA01 and the low pathogenic ISAV strain RPC/NB-04-085-1, both methods showed a 100-fold lower detectable titer for RPC/NB-04-085-1 but with a higher number of viral RNA molecules compared to NBISA01. Overall, the SYBR Green I method overestimated copy numbers in samples having equivalent Ct values with the TaqMan probe method. Taken together, the findings suggest that the TaqMan probe method with the in vitro transcribed RNA standard curve is the preferred method for reliable and rapid quantitation of ISAV in samples.

Topics: Animals; Benzothiazoles; Diamines; Isavirus; Organic Chemicals; Orthomyxoviridae Infections; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; Staining and Labeling; Viral Load