Virus detection methods: Molecular Detection Using Nucleic Acid Probes and Polymerase Chain Reaction Assays
المؤلف:
Patricia M. Tille, PhD, MLS(ASCP)
المصدر:
Bailey & Scotts Diagnostic Microbiology
الجزء والصفحة:
13th Edition , p809-812
2025-12-21
41
During the past decade, the introduction of nucleic acid detection techniques into the clinical virology laboratory has resulted in a major shift in testing strategy. With the use of both nucleic acid detection and amplification-based systems, in conjunction with automated nucleic acid isolation techniques for sample preparation, nearly all virology laboratories have access to commercial or in-house molecular assays. These technologic improvements make it possible to generate results within 2 to 6 hours. Nucleic acid detection can be accomplished using nucleic acid probes, which are short segments of DNA that hybridize with complementary viral DNA or RNA segments. The probe is labeled with a fluorescent or chromogenic tag that allows detection if hybridization occurs. The probe reaction can occur in situ, such as in a tissue thin section; in liquid; or on a reaction vessel surface or membrane. A DNA probe test used to detect papillomavirus DNA in a smear of cervical cells is illustrated in Figure 1. Nucleic acid probes are most useful when the amount of virus is relatively abundant; viral culture is slow or not possible; and immunoassays lack sensitivity or specificity.
Fig1. Smear of cervical cells stained with probe for papillomavirus DNA. Dark-staining cells contain viral DNA. (Courtesy Children’s Hospital Medical Center of Akron, Akron, Ohio.)
DNA target fragments that are too few in number in the original specimen to be detected by probes can be amplified using molecular techniques such as PCR, a method that duplicates short DNA targets thousands to a million-fold. The PCR procedure is described in more detail in Chapter 8. The PCR reaction with ensuing amplicon identification has been automated and made very rapid. Rapid PCR testing, referred to as real-time PCR, is illustrated in Figure 2. In real-time PCR, target amplification and detection occur simultaneously in the same tube; with conventional PCR, amplification and product detection take place separately. The PCR product can be detected as it is produced; novel fluorogenic probes or fluorescent dyes are used to monitor the product as it accumulates. This requires special thermal cyclers with precision optics that can monitor the fluorescence emission from the sample wells.
Fig2. Real-time polymerase chain reaction (PCR) detection of herpes simplex virus (HSV). Black, red, and light green lines represent three different HSV type 1 (HSV-1) viruses. Pink and dark green lines represent two different HSV type 2 (HSV-2) viruses. A, Cycle crossover detection of HSV-1 and HSV-2 amplicons, with all viruses detected between cycles 34 and 40. B, Melt curve confirmation of the presence of HSV-1 and HSV-2 viruses. HSV-1 amplicons melt at approximately 54°C (three HSV-1 viruses confirmed), and HSV-2 amplicons melt at approximately 68°C (one HSV-2 virus confirmed).
The PCR test can be used to amplify and detect RNA viruses by enzyme reverse transcriptase (RT). The first step in RT-PCR includes making a complementary DNA strand of the RNA segment in question. The usual PCR steps used to multiply the DNA target are then per formed, leading to DNA amplicons that, when identified, signify the presence of the original RNA sequence. The rapid appearance and broad application of molecular diagnostics require the introduction and use of standardized materials and external quality control programs. In addition, the use of universal internal controls throughout the procedure ensures accuracy. In addition, several new multiplex assays and microassays capable of detecting multiple viruses in a single reaction have been developed. These assays are particularly useful for the diagnosis of respiratory pathogens. Finally, isothermal amplification reactions, are becoming more popular.
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