REVIEW ARTICLE

New Technology in the Diagnosis of viral Disease PCR (Polymerase chain reaction) - Review
Ganapathy T1, Kumarakrishnan V B2
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ReaderDepartment of Oral and Maxillofacial Pathology, Vivekanandha Dental College & Hospital, Namakkal Dt. Professor & Head Department of Oral & Maxillofacial Pathology, Vivekanandha Dental College & Hospital,Namakkal Dt. Address for correspondence: Ganapathy T, Reader, Department of Oral & Maxillofacial Pathology, Vivekanandha Dental College & Hospital, Namakkal Dt.
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ABSTRACT: Molecular Biology is changing the face of diagnostic medicine, and infections disease of the oral soft tissues are among the targets of these advances in biotechnology. The polymerase chain reaction is a new developed molecular biology technique that can significantly amplify DNA or RNA. This powerful method has numerous application in diagnostic pathology especially in the fields of microbiology , Forensic Science, and hematology. The PCR may be used to directly detect viral DNA which may facilitate the diagnosis of viral disease. PCR amplification of DNA allows detection of specific sequences from extremely small samples. Key words: Polymerase chain reaction, Gene amplification, pathology.

Diagnostic

Introduction: Several virus are known to infect the oral mucosa. Some are established causes of disease and yet some although common in oral mucosa rarely causes identifiable disease. The possibility that viruses may be associated with or be the etiologic agents in some oral malignancies is frequently raised. The clinical recognition of viral disease in oral cavity is difficult because of similarities in the clinical picture of several mucosal disease unrelated to viral agents1. Molecular biology has recently brought about profound changes in dental research, resulting in a level of understanding of genetic, infectious and neoplastic disease. One aspect of this development is the ability to diagnosis disease on the molecular level. The diagnosis of viral infectious disease affecting the orofacial region has benefited greatly from the detection of viral DNA with exquisite levels of sensitivity2. Molecular anatomy of DNA: Chromosomal DNA transfers and stores information regarding the structure and function of the cell. The structure of DNA reflects how that information is transferred from cell to cell3. When cell cycles or divide each chromosome is so tightly bundled by histone protein. This packaging is necessary to accurately separate the duplicated chromosome between the dividing cells without physically damaging the DNA within. Once the chromosomal DNA is separated between the 2 cells, the substructures of the chromosomes relax and become

invisible on light microscopy4. The DNA within chromatin is wrapped in complexes called solenoids and nucleosomes. The information is stored in the DNA itself by 4 coding elements or nucleotides, adenine, guanine, cytosine and thymine. The 23 pairs of chromosomes contain 3 billion pairs of nucleotides. Although chromosomal DNA is packaged in tightly ordered and physically distinct structures and substructures, the information encoded is grouped by functional unit called genes5. Until recently it was believed that there were 1,00,000 genes contained in the 23 human chromosomes. Early finding from the Human genome project estimate that there are actually far fewer, approximately 35,000 to 45,000 human genes. Each gene is subdivided into structure based on function properties. The open reading frame (or message containing region) contains introns and exons. Introns are spacers than have an unclear functional significance. Exons contain the nucleotide sequences that are eventually transcribed. into Messenger RNA (RNA) and translated into protein. Situated at the start of the open reading frame is the promoter. Gene promoters contain groups of nucleotides used as binding site for proteins that start MRNA transcription. Although promoters act as On / Off switches, that activity is modulated by another important functional group of nucleotides called enhancers and repressors. Gene enhancers / repressors can be situated next to promoter or on the other side of the open reading frame (or both) and serve to elevate or suppress gene activity6.

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New Technology in the Dagnosis of viral Disease

Ganapathy T & Kumarakrishnan VB

PCR Review: PCR is initiated by the separation of double helical DNA into two single strands by being heated to 90-1000C. This is followed by annealing of synthesized oligonucleogide primers targeted to the region of interest at a lower temperature based upon the melting point, or dissociation temperature of the primers. Nucleotide bases are added to these primers in a 5' to 3' direction by a thermostable DNA polymerase to complete two identical copies of the original DNA. This process is repeated for 30 to 35 cycles - with newly synthesized extension products serving as templates for subsequent denaturation resulting in exponential amplification of a specific, defined region of the original DNA template. Several factors are important for the successful application of PCR as a diagnostic tool. Appropriately designed and carefully synthesized oligonucleotide primer are critical. Commonly 15 to 30 bases in length, a pair of well designed primer will have approximately equal length and G-C content, which facilitates the matching of appropriate annealing temperature. The PCR reaction is carried out in a buffer containing of KCI, Tris, MgCl2 and gelatin. With a denatured single strands of DNA as a template, DNA polymerase adds nucleotide bases sequentially in a 5' to 3' direction to the annealed primer. Rapid development in automation of the procedure followed by the use of thermostable DNA polymerase produced by the ubiquitous thermophilic bacterium. Thermus equaticus (Taq). Other DNA polymerases exhibiting increased stability and enhanced accuracy at higher temperature are now available. PCR products are routinely visualized on ethidium-bromide stained agarose gels, which makes the process easily adaptable to a variety of Laboratory Setting. Addition means of detection include immobilization of products on filter for southern analysis with radiolabeled a probe which usually increases the sensitivity significantly. A double stranded amplification product of good quality can also be sequenced for addition verification. As with any technique used for diagnosis, issues of sensitivity and specificity are extremely important. Largely due to efficient amplification of even trace amount of DNA it is difficult to overestimate the sensitivity or limit of detection of PCR. For example the amount of strating DNA template can be as low as a picogram (10-12g). Measure of PCR sensitivity have not been strandized but relate mostly either to amplification from serial dilution of a purified template and / or comparisons to existing diagnostic methods. The exquisite sensitivity of PCR as a diagnostic tool must be appreciated in the context of susceptibility to contamination. Recommendation for minimizing contamination and the production of false positive include.

a) Physical isolation of PCR preparations and products b) Separate and fully dedicated supplies and instruments c) Routine sterilization of applicable reagents and disposable supplies d) Routine use of gloves e) Frequent centrifuging to minimize contamination of lids by splashing f) The use of positive displacement pipettes with ejectable tips and plungers g) Premixing of reagents h) The addition of template as late as possible to minimize manipulation. i) Continuous use of positive and negative control. In general the specificity of the reaction can be altered through manipulation of two variables the annealing temperature and the magnesium concentration. Lower annealing temperatures favour non specific primer binding which results in undesirable products and increased noise to signal ratio. The optimal magnesium concentration varies, but an increase generally result in a dramatic increase in amplification efficiency of non specific 2 products . PCR Applications In The Diagnosis Of Bactrial & Viral Infections: Mycobacterium tuberculosis: The development of the polymerase chain reaction (PCR) has the potential to offer rapid and sensitive identification of pathogens without the need for culture. DNA amplification and detection can be performed in one working day. Theoretically a single organism in a clinical sample can be detected, but in practice the limit is usually around 10-1000 organisms. The most commonly used target for detection of M. Tuberculosis has been the insertion sequence IS 6110. This sequence is present up to 20 times in the M. Tuberculosis chromosome and is specific for the M. Tuberculasis complex. Other target DNA sequences used for PCR amplification to identify mycobacteria include the 65 kD heat shock protein gene (the first candidate used for PCR amplification). the MPB 64 gene and the 16 Sribosomal RNA (rRNA) gene DNA amplification by PCR for IS61 1100 performs well against the gold standard of conventional culture. For example, in one study, the sensitivity, specificity and positive predictability values for PCR were 83.5%, 99.0% and 94.2% respectively. Most studies on large collections of samples from patients with both pulmonary and extrapulmonary disease (de Lassence et al, 1992) obtained broadly similar results, placing PCR somewhere in between culture and microscopy for sensitivity. Some
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New Technology in the Dagnosis of viral Disease

Ganapathy T & Kumarakrishnan VB

studies have demonstrated that tuberculosis can be diagnosed by PCR analysis of peripheral blood7 . Herpes Simplex Virus Types I, II: PCR is able to detect HSV1, HSV2, and VZV DNA. Therefore, it is helpful in diagnosing and differentiating occult HSV, atypical presentations of HSV (such as zoster form HSV or disseminated HSV), and zoster form HSV or disseminated HSV), and unusual manifestations of VZV (such as nondermatomal, disseminated VZV or zoster sine herpete). A recent study demonstrated that PCR was a reliable method for detecting HSV and VZV1 DNA sequences from either single stained or unstained Tzanck smear preparations. The use of unstained smear provided a higher yield of detection for both of the viral infections. PCR reaction detected DNA sequences of HSV and VZV in 83% and 97% of unstained smears, respectively. In a comparison of PCR, the Tzank smear preparation, and the viral culture in detection of HSV and VZV infection, the investigators concluded that PCR was clearly superior to viral culture in identifying VZV infection and equivalent to conventional culture techniques in identifying cases of HSV and VZV infection, the investigators concluded that PCR was clearly superior to viral culture in identifying VZV infection and equivalent to conventional culture techniques in identifying cases of HSV. Another study also demonstrated that PCR was much more rapid, highly sensitive, and specific ( as compared with virus isolation) for the diagnosis of acute VZV infections8. Human immunodeficiency virus: The use of PCR in infections diseases has primarily centered on the diagnosis of viral diseases. This could have tremendous diagnostic potential in cases in which culture is difficult and time consuming or if increased sensitivity is desired. PCR offers particular promise in acquired immune deficiency syndrome (AIDS) research, where it is possible to detect PCR-amplified HIV-1 genomes from peripheral blood cells of seropositive patients. This technique may help to resolve indeterminate Western blot results. Furthermore, PCR already has been applied to the diagnosis of HIV infections in infants born to seropositave mothers. In such cases, PCR obviates the problem of serologic false positive results that result from passage of maternal antibody. Polymerase chain reaction amplification has also been used to detet human T-cell lymphotrophic virus. typeI (HTLV-I). Epstein-Barr virus (EBV). hepatitis B Virus,

cytomegalovirus (CMV), and human papillomavirus (HPV) The latter two have also been detected by PCR with the use of archival material from paraffin embedded tissue. As long as a characteristic DNA sequence is known, then detection of any virus by PCR techniques is distinctly possible. In forensic sciences. it has been possible to generate DNA sequences from extremely small samples, such s single human hairs or blood spots, which may then be used for analysis. Summary: Recent advances in molecular biology have opened a new frontier for diagnostic pathology. Although the application of recombinant DNA technology, including PCR, to assess questions in anatomic / clinical pathology is in its early stages, progress in this area has been made. Gene rearrangements and in situ hybridization analysis have already made an impact on diagnostic pathology. The application of PCR with "designer" primers used to amplify gene and gene products of interest will add to this growing list of tools to enhance present diagnostic techniques9. This article has reviewed the development and application of PCR to the diagnosis of several important viral infections affecting the head and neck region. Continued use of this powerful diagnostic tool promises substantial gains in knowledge of these diseases over the next several years. References:
1. 2. 3. 4. Syrjnen S. Viral infections in oral mucosa. Scand J D e n t R e s . 1 9 9 2 ; 100(1):17-31. Rodu B. New approaches to the diagnosis of oral soft-tissue disease of viral origin. Adv Dent Res. 1993; 7(2):207-12. Todd R, Donoff RB, Wong DT. The chromosome: cytogenetic analysis and its clinical application. J Oral Maxillofac Surg. 2000; 58(9):1034-9. Robert JW. Gene Structure and Function. In: Watson JD, Baker TA , Bell SP, Gann A , Levine M , Losick R, eds. Molecular Biology of gene. 5th ed. Menlopark CA: Cummings B; 1987:617. Todd R,Donoff RB, Kim Y, Wong DT. From the chromosome to DNA: Restriction fragment length polymorphism analysis and its clinical application. J Oral Maxillofac Surg. 2001; 59(6):660-7. Hill PJ, Stewart GS. The polymerase chain reaction in molecular and micro-biology. Biotechnol Genet Eng Rev. 1992;10:343-77. Kim Y, Flynn TR, Donoff RB, Wong DT, Todd R. The gene: the polymerase chain reaction and its clinical application.J Oral Maxillofac Surg 2002;60(7):80815. Marshall BG, Shaw RJ. New technology in the diagnosis of tuberculosis. Br J Hosp Med. 1996;55(8):491-34. Cohen PR. Tests for detecting herpes simplex virus and varicella-zoster virus infections. Dermatol Clin. 1994;12(1):51-68. Remick DG, Kunkel SL, Holbrook EA, Hanson CA. Theory and applications of the polymerase chain reaction. Am J Clin Pathol.1990; 93 (4 Suppl 1):S49- 54.

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