Nested PCR

Detection of HHV-6, EBV and HTLV-2 Genomic DNAs by Nested PCR

[For more information about these tests click here.]

Over the past several months, the Institute for Viral Pathogenesis has developed a number of new molecular diagnostic technologies. They are being used in our current studies. These sophisticated new “nested PCR” systems are significantly more sensitive than other currently available PCR technologies.

A schematic of nested PCR is shown in the figure below. (Scroll down to view figure.)

Nested PCR uses two sets of amplification primers. The target DNA sequence of one set of primers (termed “inner” primers) is located within the target sequence of the second set of primers (termed “outer” primers). In practice, a standard PCR reaction is first run with the patient sample using the “outer primers”. Then a second PCR reaction is run with the “inner primers” using the product of the first reaction as the amplification target. This procedure increases the sensitivity of the assay by reamplifying the product of the first reaction in a second reaction. The specificity of the assay is increased because the inner primers amplify only if the first PCR reaction yielded a specific product.

HHV-6 Specific Nested PCR System 
The primers in this PCR system target the HHV-6 U54 gene (Gompels et al, 1995)., Use of the outer primer set, which recognize both the A and B variants of the virus equally in the clinical diagnostic setting has been described previously (Drobysky et al, 1994). The outer PCR primers produce a PCR product that is 383 base pairs in length. Two separate sets of inner primers are used in this system. One set recognizes only the A variant of HHV-6 , while the other set recognizes only the B variant of the virus. Both sets of inner primers produce a product that is 200 base pairs in length.

The PCR product is visualized by agarose gel electrophoresis and ethidium bromide staining. In titration experiments using normal human serum spiked with a plasmid containing the PCR target sequence, it was found that this system could detect less than 10 HHV-6 genomes per milliliter of serum (approximately 2 viral genomes per PCR reaction). We have used this PCR system to detect HHV-6 DNA in the sera of patients with multiple sclerosis, chronic fatigue syndrome and liver transplant recipients.

EBV Specific Nested PCR System
This PCR system targets the EBV gene encoding the viral latent membrane protein one (LMP-1) and detects the two types of EBV equally as assessed using the prototype B95-8 and AG876 strains for type 1 and type 2 viruses, respectively (Kieff and Rickinson, 2001; Sample et al, 1994). The product of the outer primers in this system is 407 base pairs in length while the product of the inner primers is 243 base pairs long. This PCR system has been successfully used to detect EBV DNA in the sera of patients with chronic fatigue syndrome.

HTLV-2 Specific Nested PCR System
Human T Cell Lymphotropic Virus Type 2 (HTLV-2) has been implicated as being involved in some cases of chronic fatigue syndrome (DeFreitas et al, 1991). The Institute has recently been awarded a grant to attempt to confirm this earlier work. In pursuit of this study a nested PCR primer system targeting the viral gag gene has been designed to detect HTLV-2 DNA in patient blood leukocyte samples. The product of the outer primers of this system is 468 base pairs in length while the product of the inner primers is 206 base pairs in length. The figure below shows an ethidium bromide stained agarose gel analyzing a titration of the sensitivity of this PCR system. Serial ten-fold dilutions of a positive control preparation of a plasmid containing the target HTLV-2 DNA were analyzed by PCR with the outer primers alone and with the combined nested PCR primers.

As is evident in the figure, the outer primers were able to detect the viral DNA down to a 10-5 dilution. In contrast, the full nested PCR system detected the viral DNA down to a 10-8 dilution. Thus, the nested system is approximately 1000 times more sensitive than the single primer system.
References Gompels UA, Nicholas J, Lawrence G, Jones M, Thomson BJ, Martin ME, Efstathiou S, Craxton M and Macaulay HA. The DNA sequence of human herpesvirus?6: structure, coding content, and genome evolution. Virology 1995; 209:29?51.

Drobyski WR, Knox KK, Majewski D and Carrigan DR. Fatal encephalitis due to variant B human herpesvirus 6 infection in a bone marrow transplant recipient. N Engl J Med 1994; 330:1356?1360.

Kieff E and Rickinson AB. Epstein-Barr virus and its replication. Fields Virology, Fourth Edition. DM Knipe and PM Howley (eds.) Lippincott Williams and Wilkins. Philadelphia, Pennsylvania. 2001. pp. 2511-2573.

Sample J, Kieff E and Kieff E. Epstein-Barr virus types 1 and 2 have nearly identical LMP-1transforming genes. J Gen Virol 1994; 75:2741-2746.

DeFreitas E, Hilliard B, Cheney PR, Bell DS, Kiggundu E, Sankey D, Wroblewska Z, Palladino M, Woodward JP AND Koprowski H. Retroviral sequences related to human T-lymphotropic virus type II in patients with chronic fatigue immune dysfunction syndrome. Proc Natl Acad Sci 1991; 88:2922?2926.

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