Single-tube amplification method for high-throughput screening of HSV
Cindy Walker-Peach Beti Belachew Peter Pingerelli
Melanie Palmer Dwight DuBois
Stratagene introduces the HSVision molecular beacon detection module,*,,ff a single-tube method to quickly detect HSV (herpes simplex virus) DNA. Because it is performed in a closed-tube system and requires no postamplification manipulation, HSV detection is simplified, and the risk of contamination is markedly reduced. Using molecular beacon technology, this detection module includes an HSV molecular beacon that hybridizes to both HSV-1 and HSV-2 amplicons, a molecular beacon specific for an internal amplification control (IAC), a mixed HSV-1 and HSV-2 DNA standard, target-specific DNA primers, and an optimized PCR buffer. This module can be easily adapted for high-throughput screening
HSVs have worldwide distribution and cause a wide variety of illnesses in humans. Since this virus remains latent in the host for life, it can be reactivated to cause lesions at or near the site of initial infection.1 Most common are mucocutaneous infections, which primarily affect oral-facial and genital surfaces. However, more serious HSV infections can also involve the central nervous system and viscera, particularly in newborns and immunocompromised patients, where they are often life threatening. There are two HSV subtypes, HSV-1 and HSV-2,2 which are serologically and genetically distinct. HSV-1 and HSV-2 share a similar overall genomic structure and contain approximately 50% nucleotide sequence identity.
Current diagnostic methods for HSV infection include serological testing, viral culture, and more recently, PCR detection.3 Stratagene has developed a single-tube, real- time PCR format, which provides rapid and sensitive detection with high-throughput capability, and significantly increase the speed, throughput, and reliability of HSV detection.
Molecular beacons are single-stranded oligonucleotides that possess a stem-and-loop hairpin structure.4-7 The loop is comprised of a DNA sequence complimentary to the target DNA, and the stem is formed by short complimentary sequences at opposite ends of the molecule. The ends of molecular beacons are labeled with a fluorophore and a quencher, respectively. When the molecular beacon is folded, the fluorophore and quencher are in close proximity, and fluorescence is quenched. However, when the molecular beacon is bound to a complimentary target, the fluorophore and quencher are separated, and the molecular beacon fluoresces (Figure 1).
The HSVision module primers are derived from a 109 base-pair region of the gB gene that is highly conserved in HSV-1 and HSV-2 genomes.8 Because PCR reactions are often susceptible to a variety of inhibitors in biological specimens, an IAC is included in the HSV detection module to control for PCR failure. The IAC is a plasmid encoding a sequence identical in size and GC content to the HSV target DNA. The HSV-TC molecular beacon is labeled on the 5 end with the fluorophore tetrachloro 6-carboxyfluorescein (TET). The IAC-specific molecular beacon is labeled on the 5 end with 6-carboxyfluorescein (FAM). DABCYL is attached to the 3 end both molecular beacons and is used to quench fluorescence. The HSV-TC and IAC molecular beacons are multiplexed in a single-tube, real-time PCR reaction, allowing simultaneous detection of HSV and IAC.
Amplification is performed with a PCR mixture that contains the HSV-TC and IAC molecular beacons, HSV PCR primers, dNTPS, Taq2000 DNA polymerase, an IAC DNA template, and an optimized PCR buffer. PCR is performed in a real-time spectrofluorometric thermal cycler. The presence of HSV-1 or HSV-2 DNA is determined by comparing the endpoint fluorescence value and/or the threshold cycle (Ct) values of the HSV DNA to the IAC control. As shown in Figure 2, 105 to 102 copies of HSV-1 (Figure 2) and HSV-2 (Figure 2) are reliably detected with the HSV-TC molecular beacon.
Stratagenes single-tube HSVision molecular beacon detection module offers several advantages over existing HSV screening techniques: The test is performed in a closed tube so no post-PCR manipulation of samples is required. Because the entire reaction takes place in one tube, the test saves time and effort and significantly reduces the risk of PCR product carry-over contamination. Furthermore, this multiplexed assay includes both an HSV-specific molecular beacon as well as an internal control so assay performance and validation are realized. Hence, a true negative result can be discriminated from a false negative result due to PCR failure. In addition, the inclusion of both high- and low-copy number mixed HSV-1/HSV-2 DNA standards permits assay sensitivity to be validated. Using a 96-well format, samples can be completely screened in approximately 4 hours.
* Patents pending