Diagnostic Virology Protocols by John R. Stephenson, Alan Warnes

By John R. Stephenson, Alan Warnes

A set of state of the art concepts for detecting lots of the significant viruses that afflict mankind, together with influenza, hepatitis, herpes, polio, mumps, HIV, and lots of extra. The concepts are well-tested, simply reproducible, and with ease hire the entire new technologies-PCR, RIA, ELISA, and latex-agglutination-that have revolutionized the sphere. those tools not just give the opportunity to do the required research in hours rather than days, yet can be automatic in a laboratory havng in simple terms low degrees of organic containment. often, the protocols for viruses inflicting human ailments might be tailored to comparable viruses of veterinary significance. via its state of the art tools a doctor can, for the 1st time, ascertain early in a viral an infection which antiviral drug could be used and reduce the interval of therapy to prevent pointless negative effects.

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And Quirke, P. (1990) Tissue extraction of DNA and RNA and analysis by the polymerase chain reactton. J Clin. PathaI 43,499-504 66 Lau, J. , Qian, K. , Wu, P. C , and Davis, G. L. (1993) Rtbonucleotide vanadyl complexes inhibit polymerase chain reactton. Nucl Aczds Res 21,2777. 5 Rapid Detection and Identification of Dengue Viruses by Reverse Transcriptase/Polymerase Chain Reaction Dennis W. Trent, Gwong-Jen Chang, A. Vance Vorndam, and Robert S. Lanciotti 1. Introduction Introduction of the polymerase chain reaction (PCR) technique as a rapid and sensitive method for amplification of DNA has resulted m development of new specific nucleic acid-based techniques for clmical diagnosis of infections (I).

Followmg amplitication of enterovirus RNA, the nested PCR protocol described here usually gives rise to a single band of approx 148 bp that comtgrates with the 142/154 doublet band of Life Technologies’ kb DNA ladder, although a smaller band corresponding to primer dimers is usually present. Occastonally larger bands are also seen, which may be due to PCR product carried over from the first PCR cycle, or seminested PCR products We do not consider Southern blot hybridization to be necessary to confirm posmve PCR results After continuous use of this assay for 3 yr in this laboratory, no sample has ever gtven rise to a PCR product of correct size that was not subsequently confirmed to be of enterovirus ortgm by nucleottde sequence analysis or reamplification using alternative primers The specificity of nested PCR amplification, which is dependent on four specific primer/target interactions, can thus be assumed to be equal to or greater than that of single round PCR followed by Southern blot hybrtdtzatton, which 1sdependent on only three specific primer/target or probe/ target interactions References 1 Melnick, J.

Infectious virus can be isolated from serum during the acute phase of DEN (8) and YF virus infections. Flaviviruses can be isolated from infected mosquitoes, blood/serum or autopsy samples by maculation of mosqutto or mammalian cell cultures, suckling mice, or mosquitoes (2). Amplification of the flavivirus RNA genome by PCR is a two-step process which first requires converston of the genome target to DNA by the enzyme reverse transcriptase and amplification of the target by repetitive cycles of oligonucleotide primer (amplimer)-directed DNA replicatton (9-15).

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