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2000, Number 3

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Rev Inst Nal Enf Resp Mex 2000; 13 (3)

Incorporation of the RT-PCR technique to the detection of respiratory syncitial virus.

Archundia SFJ, Alejandre CJE, Cabello GC, Rosete ODP, Manjarrez ZME

Language: Spanish
References: 17
Page: 145-152
PDF size: 395.37 Kb.


ABSTRACT

Objective: To standardize and incorporate the molecular biology technique of RT-PCR to the detection of respiratory syncitial virus (RSV) to complement and enrich the dignosis. Material and methods: RSV reference strains from groups A and B (virus stock ATCC) were propagated and titered with HEp-2 cells. Indirect immunofluorescence and RT-PCR tests were performed. For this, RNA was extracted with trizol and cDNA was obtained by retrotranscription. For the PCR, oligonucleotides were obtained which amplify the fragment of the gene coding for protein G of RSV. To test specificity, viruses from the same family, measles and parainfluenza, and of different families, influenza and adenovirus, were used. Sensitivity was tested with several dilutions of viral cDNA. Results: The cytopathic effect may be clearly observed from the fourth to the eighth day. Immunofluorescence is, as known, a sensitive and specific test for the virus. The RT-PCR was effective for the amplification of cDNA and also proved to be so for RS, since it does not amplify genetic material of other viruses, oven if they belong to the same family. The sensitivity of the test is high; it amplifies amounts as small as picograms of cDNA. Conclusion: The speed of immunofluorescence may be used to give a presumptive diagnosis which can be confirmed by isolation and propagation of the virus in cell culture. The RT-PCR technique with the oligonucleotides used was sensitive, specific and quick, and can therefore be used to support the clinical diagnosis. This does not imply that previous techniques should be substituted but may it does add to the options and gives support to the diagnosis of viral infections.


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