Biopolym. Cell. 1991; 7(3):31-35.
http://dx.doi.org/10.7124/bc.0002CB
Polymrase Chain Reaction (PCR) for the diagnosis of virus infection: human cytomegalovirus
1Vinogradskaya G. R., 1Goryshin I. Yu., 2Novikova L. N., 3Plutalov O. V., 2Bashmakova M. A., 3Berlin Yu. A., 4Tsinzerling V. A., 3Schwartz E. I., 1Lanzov V. A.
  1. B. P. Konstantinov Institute of Nuclear Physics, Academy of Sciences of the USSR
    Gatchina, Leningrad distr., USSR
  2. Institute of Obstetrics and Gynecology, Academy of Medical Sciences of the USSR
    Leningrad, USSR
  3. M. M. Shemyakin Institute of Bioorganic Chemistry, Academy of Sciences of the USSR
    Moscow, USSR
  4. Leningrad Pediatric Medical Institute, Ministry of Health of the RSFSR
    Leningrad, USSR

Abstract

A rapid, sensitive and precise procedure of detecting cytomegalovirus (CMV) DNA in human tissue and biological liquid by means of the thermostable DNA-polymerase from Thermus thermophilus is described. The procedure consists of two consecutive PCRs. The-first one includes 40 cycles of denaturation, primer annealing and chain extension with two 20-base primers chosen from the conserved sequence ot MIE (major immediate early) gene of CMV. The second reaction includes from 20 up to 50 cycles consisting of the same steps but with 20-base primers chosen to amplify the internal part of the fragment amplified during the first reaction. The final fragment is detected by the ethidium bromide staining after polyacrylamide gel electrophoresis. Verification of the band includes restriction enzyme analysis. Use of pCM5018 plasmid bearing the CMV MIE gene shows that the proposed procedure can detect as little as several (from 1 to 5) molecules of DNA.
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References

[1] Samohin PA. Cytomegalovirus infection in children. M.: Medicine, 1987. 161 p.
[2] Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985;230(4732):1350-4.
[3] Demmler GJ, Buffone GJ, Schimbor CM, May RA. Detection of cytomegalovirus in urine from newborns by using polymerase chain reaction DNA amplification. J Infect Dis. 1988;158(6):1177-84.
[4] Li JM, Morgan MA, Rascon D. et al. Polymerase chain reaction for detection of human cytomegalovirus. Abstrs Ann Meet Amer Soc Microbiol. New Orlean, 1989; S-10:372.
[5] Fiss E, York MK, Brooks GF. Detection of cytomegalovirus in bronchial lavage and blood specimens by means of the polymerase chain reaction. Abstrs Ann Meet Amer Soc Microbiol. New Orlean, 1989; C-176:422.
[6] Forman M, Charache P, Arthur R. The use of polymerase chain reaction for detection of cytomegalovirus in clinical specimens. Abstrs Ann Meet Amer Soc Microbiol. New Orlean, 1989; D-238:122.
[7] Rosenberg E, Keiser J, Gross J. et al. Use of the polymerase chain reaction technique to detect cytomegalovirus. Abstrs Ann Meet Amer Soc Microbiol. New Orlean, 1989; D-239:122.
[8] Vinogradskaya GR, Goryshin IYu, Plutalov OV. Using polymerase chain reaction for detection of cytomegalovirus in human tissues. VII Proc. symposium. "Mol. mechanisms genet. processes" M., 1990:2037.
[9] RГјger R, Bornkamm GW, Fleckenstein B. Human cytomegalovirus DNA sequences with homologies to the cellular genome. J Gen Virol. 1984;65 (Pt 8):1351-64.
[10] Goelz SE, Hamilton SR, Vogelstein B. Purification of DNA from formaldehyde fixed and paraffin embedded human tissue. Biochem Biophys Res Commun. 1985;130(1):118-26.
[11] Davis RW, Botstein D, Roth JR, A Manual for Genetic Engineering, Advanced Bacterial Genetics. Cold Spring Harbor Laboratory. 1980; 251 p.
[12] Shvarts EI, Kaboev OK, Gol'tsov AA, Vinogradov SV, Lebedenko EN. Primer-dependent amplification of 2 sites of human beta-globin gene. Bioorg Khim. 1988;14(11):1577-9.
[13] Stenberg RM, Thomsen DR, Stinski MF. Structural analysis of the major immediate early gene of human cytomegalovirus. J Virol. 1984;49(1):190-9.