Elsevier

The Lancet

Volume 335, Issue 8705, 30 June 1990, Pages 1598-1599
The Lancet

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Detection by PCR of HHV-6 and EBV DNA in blood and oropharynx of healthy adults and HIV-seropositives

https://doi.org/10.1016/0140-6736(90)91433-BGet rights and content

References (4)

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    Human herpesvirus-6 is closely related to human cytomegalovirus

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  • R. Baer et al.

    DNA sequence and expression of the B95-8 Epstein-Barr virus genome

    Nature

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There are more references available in the full text version of this article.

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    Extracted DNAs were assessed for their suitability for PCR analysis by a control reaction designed to amplify a fragment of the 407-base paired (bp), of the β-globin gene as described previously [33]. The presence of EBV was investigated by a PCR protocol using a specific set of primers EBV-S (3′-AACATGCTGTATGCCTCGCAGCG-5′) and EBV-R (3′-AATTACTGGCGTGAATTGTGCCCA-5′) amplifying a 176-bp sequence in the BamHI G region of the EBV genome [15]. The PCR reactions were performed using 200 ng of DNA template in a total volume of 25 μl, containing 0.2 μM of each sense and antisense primers, 10 mM Tris–HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 mM of each dNTP, and 1 U of Taq DNA polymerase (Promega, Madison, USA) in a PTC 200™ DNA engine thermal cycler (MJ Research, Watertown, USA).

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    After discharge, they were followed by a child neurologist for 4 to 7 years. Total DNA was extracted by phenol-chloroform from 100 μL of cerebrospinal fluid and amplified in a 100-μL reaction mixture containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl, 0.01% (w/v) gelatin, 200 μM each of the four deoxyribonucleoside triphosphates, 2.5 units of AmpliTaq Gold polymerase (Applied Biosystems, Foster City, CA), and 50 pmol of both primers [21], chosen from the U67 gene (5′-AAG CTT GCA CAA TGC CAA AAA ACA G-3′ and 5′-biotin-CTC GAG TAT GCC GAG ACC CCT AAT C-3′). The target sequence was amplified with the following thermal profile: step 1, 95°C for 10 minutes; step 2, 95°C for 30 seconds, 51°C for 30 seconds, and 72°C for 1 minute, for 40 cycles; and step 3, 72°C for 10 minutes.

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