2Department of Zoology, University of Oxford, Oxford, UK
3GeneFirst Ltd, London, UK
4Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, USA
Quantitative PCR assays are now the standard method for viral diagnostics. These assays must be specific, as well as sensitive, to detect the potentially low starting copy number of viral genomic material. We describe a new technique, polymerase chain displacement reaction (PCDR), which uses multiple nested primers in a rapid, capped, one-tube reaction that increases the sensitivity of normal quantitative PCR (qPCR) assays. Sensitivity was increased by approximately 10-fold in a proof-of-principle test on dengue virus sequence. In PCDR, when extension occurs from the outer primer, it displaces the extension strand produced from the inner primer by utilizing a polymerase that has strand displacement activity. This allows a greater than 2-fold increase of amplification product for each amplification cycle and therefore increased sensitivity and speed over conventional PCR. Increased sensitivity in PCDR would be useful in nucleic acid detection for viral diagnostics.
PCR is a key technique used in molecular biology to amplify specific regions of DNA; the amplified product is traditionally detected by gel electrophoresis. The emergence of quantitative PCR (qPCR) using fluorescent probes or dyes now allows amplification and analysis simultaneously, conferring rapid detection and quantification of target DNA. qPCR also enables higher throughput with a reduced risk of cross-contamination, as there are no post-PCR manipulations. The amplified product in qPCR can be detected by either nonspecific DNA binding dyes, for example SYBR green, which binds to any double-stranded DNA present, or by probes that hybridize to specific target sequences (1-4).
In virus detection, qPCR has now become the standard technique, as it is quick, sensitive, and easier to perform than the more traditional method of culturing the virus in cell lines (5). Virus culture can take up to 2 weeks, depending on the virus, and the result can be hindered by the presence of antibodies in the patient's serum. qPCR is also being used over serological analysis, which detects antibodies specific to the virus in a patient's serum. Serological analysis prevents early, rapid diagnosis, as antibodies are generally only produced 6–14 days after infection. Serological analysis is also not possible when trying to detect the presence of virus in samples that are not human serum, for example in arboviral vectors such as mosquitoes. Nucleic acid detection with qPCR has a further advantage of quantifying the copy number of virus in a patient's plasma, which can be vital for monitoring some diseases (6).
Conventional PCR uses a pair of primers and therefore produces a maximum of a 2-fold increase in amplicon per amplification cycle. We have developed a modification to PCR that uses more than one pair of primers; we call this variant polymerase chain displacement reaction (PCDR). Nested primers are designed that flank the region of interest; when the outer and inner primers are extended, the extended strand of the outer primer causes displacement of the extended strand produced from the inner primer (Figure 1). The polymerase used in PCDR is a modified Taq DNA polymerase that possesses strand displacement activity and lacks 5′ to 3′ exonuclease activity; therefore degradation of the inner primer extension product does not occur. In PCDR, we use a dual-labeled probe, which has a quencher at the 3′ end and a fluorophore at the 5′ end. When there is no target present, the two ends of the probe come close to one another, in a random coil, resulting in the fluorophore being quenched; once there is template for the probe to bind to, the quencher and fluorophore are separated, allowing fluorescence to be generated (7). PCDR allows more than two amplicons to be produced after each amplification cycle and therefore greater sensitivity and increased speed of assay. An increase in the sensitivity of amplification assays would be beneficial in diagnostic uses for virus detection where there is a low starting copy number or where sensitivity is lost by PCR inhibitors present in crude extracts (8).
Another advantage of using multiple primers in PCDR for viral detection is due to the high frequency of mutations that occurs in viral genomes, producing multiple subtypes with a high level of genetic variation within each virus (9, 10). Primers are very specific; therefore detection via PCR is vulnerable to false negatives due to genome sequence diversity. In viral diagnostics, PCR primers need to be designed so that all subtypes are identified and new subtypes could possibly be recognized. In one study, four commercial quantitative viral load assays for HIV were tested with a panel of diverse patient samples taken from multiple regions and as few as 88.6% were positively detected from 97 plasma samples. These false negatives were found to be due to mutations in the primer or probe binding sites (6). A single pair of primers is more susceptible to false negatives due to these mutations, whereas if four primers are used together, as in PCDR, it is much less likely that mutations would occur in both primer binding sites and therefore amplification would still occur.
To determine if sensitivity and speed could be improved by PCDR in viral detection, we developed an assay to identify the presence of dengue virus sequence. Dengue virus (DENV) is a positive strand RNA flavivirus that has four distinct serotypes (DENV1–4) and is the causative agent of dengue fever (11). Dengue is a mosquito-borne disease that is endemic in more than 100 countries, with an incidence that is rapidly increasing (12-15). While dengue fever presents with flu-like symptoms, this can progress to dengue hemorrhagic fever, which can be fatal. This usually occurs if the patient is infected with a second DENV serotype. PCR assays have been developed for the detection of dengue viral RNA. Some of these assays are capable of serotyping and/or able to quantify the viral load, which may be an indicator of disease severity (16). Only one PCR assay for DENV has been commercialized to date (RealArt; artus/Qiagen, Valencia, CA, USA) (17). The other in-house assays developed include nested reverse transcriptase PCR, where the result is visualized by gel electrophoresis (18-20); however this is not suitable for high-throughput diagnostics, and therefore multiplex quantitative assays have also been designed, including one-step quantitative reverse transcriptase and multiplex quantitative assays (21-24).