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A polymerase chain reaction-based assay for diagnosing varicella-zoster virus retinitis. The scientist used two different types of enzymes: the Mlu I and the Ava I. The assay does not require the use of hybridization probes or radioactive reagents.
A polymerase chain reaction-based assay for diagnosing varicella-zoster virus retinitis. The scientist used two different types of enzymes: the Mlu I and the Ava I. The assay does not require the use of hybridization probes or radioactive reagents.
A polymerase chain reaction-based assay for diagnosing varicella-zoster virus retinitis. The scientist used two different types of enzymes: the Mlu I and the Ava I. The assay does not require the use of hybridization probes or radioactive reagents.
A polymerase chain reaction-based assay for diagnosing varicella-zoster
virus retinitis in patients with acquired immunodeficiency syndrome
a) What species did they use (explain why) The scientist used two different types of enzymes: the Mlu I and the Ava I. These enzymes have the function of restriction digest patterns. Restriction digests were performed in a 30-mu l volume that contained 20-mu l of polymerase chain reaction product. The digest reactions were incubated for 2 hours at 37 0 C. Purified varicella- zoster virus DNA served as a positive control for all assays. They were designed to amplify a 326-base pair (bp) DNA fragment of immediate- early (IE) 63 gene in the varicella-zoster virus genome.
b) What is the context of this research linked to the PCR, in other words, why are they using PCR. Scientist needed to nest primer sets and confirm the specificity of the polymerase chain reaction-amplified products by restriction enzyme analysis. The assay does not require the use of hybridization probes or radioactive reagents and can be completed within 24 hours. The speed with which this assay can be performed is very important because progressive outer retinal necrosis syndrome progresses at an extremely rapid rate and requires prompt therapeutic intervention.
c) Explain the methodology (use of restriction enzymes, specific primers, specific thermo cycles, what region of DNA or RNA or any genetic material did they amplify) Specificity of the amplification products was confirmed by size (326 bp) as well as by restriction enzyme analysis. Digestion of the amplified products with Mlu I gave the expected restriction fragments of 275 bp and 51 bp (lanes 4 and 8), and digestion with Ava I produced the expected restriction fragments of 221 bp and 105 bp (lanes 5 and 9). After an initial denaturation at 94 C for 2 minutes, 40 polymerase chain reaction cycles were performed at 94 C for 30 seconds, at 55 C for 30 seconds, and at 72 C for 45 seconds.
d) Describe the conclusions they obtained regarding the use of PCR (was the problem or question solved? How did PCR make it possible?) It was developed a rapid, sensitive, and specific polymerase chain reaction-based diagnostic assay for varicella-zoster virus DNA that will assist in the diagnosis of varicella-zoster virus retinitis in patients with AIDS. No polymerase chain reaction products of the appropriate size were detected when water, rabbit skin cell, human, cytomegalovirus, or herpes simplex virus DNA was substituted for target varicella-zoster virus DNA in the polymerase chain reaction.
Resume: The purpose of this experiment was to develop a laboratory assay based on the polymerase chain reaction for the diagnosis of varicella-zoster virus retinitis in patients with acquired immunodeficiency syndrome (AIDS). For this, they developed and tested a polymerase chain reaction-based assay for the detection of varicella-zoster virus DNA in vitreous samples. The results were that varicella-zoster virus DNA was detected in 11 of 14 vitreous samples from AIDS patients. Varicella-zoster virus DNA was detected in only two of 75 control vitreous samples from immunocompetent patients with vitreoretinal disease and two of 88 control vitreous samples from patients with AIDS and vitreoretinal inflammatory disease not related to progressive outer retinal necrosis syndrome. Finally, scientist concluded that the new polymerase chain reaction-based diagnostic assay for varicella- zoster virus DNA, will assist in the diagnosis of varicella-zoster virus retinitis in patients with AIDS.
Short, G. A., Margolis, T. P., Kuppermann, B. D., Irvine, A. R., & al, e. (1997). A polymerase chain reaction-based assay for diagnosing varicella-zoster virus retinitis in patients with acquired immunodeficiency syndrome. American Journal of Ophthalmology, 123(2), 157- 64. Retrieved from http://search.proquest.com/docview/229414213?accountid=11643