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New PCR Primers for Human Diseases

Screen samples for disease states or pathogens

Gabriela M. Tobal
Stratagene Cloning Systems, Inc.

W. S. Nichols

Stratagene is introducing PCR primer sets for detecting 13 human diseases. The sequences of these primers have been published previously and are well characterized. With these tested primer sets, medical researchers can use PCR technology to identify the presence of many human diseases.

Molecular detection and genetic characterization of human disease are key components of current research in medicine. In order to detect the presence of genetic mutation and infectious agents, PCR is used routinely in research laboratories. The speed and sensitivity of PCR-based assays allow shorter detection times for bacterial and viral pathogens whose detection would otherwise require culturing and staining of samples, detecting metabolic products and performing serologic tests.

To aid in the diagnosis of specific diseases in biological materials, Stratagene is introducing a series of PCR primers that are derived from sequences published in GenBank. These human disease primers amplify genes from samples infected with the following organisms: Human immunodeficiency virus, type 1 (HIV-1),1,2 hepatitis B (HBV),3 hepatitis C (HCV) (RT-PCR primers*, use cDNA as template),4,5 herpes simplex virus, types 1 and 2 (HSV-1, HSV-2),6-8 human herpesvirus 6 (HHV-6),9,10 parvovirus B19,11 genital human papillomavirus (HPV),12-14 cytomegalovirus (CMV),15,16 Toxoplasma gondii,17 varicella-zoster virus (VZV),18 Epstein-Barr virus (EBV)19 and adenovirus.20 Primers to detect the human coagulation factor V (Leiden) mutation21 are also available.

PCR Assays of Primer Sets

Figure 1

Figure 2

Each primer set was used in PCR amplifications of DNA templates from infected serum samples, with genomic DNA from infected cells or with cDNA made from RNA isolated from infected cells. The control reactions were performed with DNA from both uninfected sera and human cells. The specificity of each primer set is shown in figure 1. Each amplification yielded a single product of the appropriate size in reactions with DNA from infected serum samples (figure 1 and figure 2), whereas amplification products were not detected in any of the negative controls, uninfected serum samples (figure 1).

Assay for the Factor V Mutation

The factor V (Leiden) mutation is associated with thrombophilia. In order to test for a point mutation of G1691A, the gene coding for human coagulation factor V, the factor V (Leiden) mutation assay is used. This assay requires an initial PCR amplification with subsequent digestion with the Mnl I restriction enzyme that yields a restriction fragment length polymorphism for the wild-type, heterozygous and homozygous genotypes (figure 2). Stratagenes primer set for performing the factor V mutation assay includes the Mnl I enzyme and the corresponding restriction enzyme buffer.

Conclusions

PCR technology is a powerful method for amplifying specific regions of DNA and can be used for detecting pathogens or mutations in DNA samples. Because PCR is capable of amplifying extremely small amounts of bacterial or viral DNA, it can be used for early detection of disease. Medical researchers can use Stratagenes Primer Sets for Human Diseases to screen DNA samples for the specified pathogens or mutations. Stratagene has packaged groups of primer sets, available at reduced rates.

REFERENCES

  1. Kwok, S., et al. (1993) In Diagnostic Molecular Microbiology: Principles and Applications, pp. 309-315. American Society for Microbiology, Washington, D.C.

  2. Kwok, S., et al. (1990) Nucleic Acids Res. 18: 999-1005.

  3. Yokosuka, O., et al. (1993) In Diagnostic Molecular Microbiology: Principles and Applications, pp. 322-326. American Society for Microbiology, Washington, D.C.

  4. Cha, T., et al. (1991) J. Clin. Microbiol. 29: 2528-2534.

  5. Wilbur, J.C., et al. (1993) In Diagnostic Molecular Microbiology: Principles and Applications, pp. 327-331. American Society for Microbiology, Washington, D.C.

  6. Cone, R.W., et al. (1993) In Diagnostic Molecular Microbiology: Principles and Applications, pp. 337-343. American Society for Microbiology, Washington, D.C.

  7. Cone, R.W., et al. (1991) J. Infect. Dis. 164: 757-760.

  8. Stuve, L.L., et al. (1987) J. Virol. 61: 326-335.

  9. Klotman, M.E., et al. (1993) In Diagnostic Molecular Microbiology: Principles and Applications, pp. 501-510. American Society for Microbiology, Washington, D.C.

  10. Buchbinder, A., et al. (1988) J. Virol. Methods 21: 191-197.

  11. Clewley, J.P. (1993) In Diagnostic Molecular Microbiology: Principles and Applications, pp. 367-373. American Society for Microbiology, Washington, D.C.

  12. Bauer, H.M., et al. (1993) In Diagnostic Molecular Microbiology: Principles and Applications, pp. 407-413. American Society for Microbiology, Washington, D.C.

  13. Bauer, H. M., et al. (1991) JAMA. 265: 472-477.

  14. Manos, M.M., et al. (1989) Cancer Cells 7: 209-214.

  15. Espy, M.J., et al. (1993) In Diagnostic Molecular Microbiology: Principles and Applications, pp. 350-355. American Society for Microbiology, Washington, D.C.

  16. Shibata, D.W., and Martin, J. (1988) J. Infect. Dis. 158: 1185-1192.

  17. Burg, L.J., et al. (1989) J. Clin. Microbiol. 27: 1787-1792.

  18. Puchhammer-Stockl, E. (1993) In Diagnostic Molecular Microbiology: Principles and Applications, pp. 356-360. American Society for Microbiology, Washington, D.C.

  19. Telenti, A. (1993) In Diagnostic Molecular Microbiology: Principles and Applications, pp. 344-349. American Society for Microbiology, Washington, D.C.

  20. Kew, O., et al. (1993) In Diagnostic Molecular Microbiology: Principles and Applications, pp. 389-393. American Society for Microbiology, Washington, D.C.

  21. Ridker, P.M., et al. (1995) N. Engl. J. Med. 332: 912-917.


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