Evaluating risky sexual behaviors and the impact of behavioral interventions is based on self-report, which is prone to reporting bias. We previously studied Y-chromosome sequence detection by polymerase chain reaction (PCR) of vaginal fluid, and hypothesized that Y chromosome sequences would not be detected in vaginal fluid without recent unprotected sexual intercourse or condom failure.1,2 We demonstrated that Y chromosome sequences were detectable for up to 2 weeks postcoitus, and furthermore, condoms were effective in preventing transmission of detectable Y-chromosome material to women (Ghanem KG-Unpublished data—submitted as companion publication).
Wide use of this technique was previously limited by relatively cumbersome laboratory methods. Furthermore, quantification using traditional PCR required use of molecular ladder standards and was also subject to substantial variability. In response, we developed and evaluated a real-time PCR assay for Y-chromosome detection using the Roche LightCycler® system, which can be easily used in settings where routine molecular testing is performed.
The research described here was approved by The Johns Hopkins University School of Medicine IRB. Initial characterization of the assay was conducted with artificially semen-spiked swabs. The swabs were exposed to different concentrations of semen by placing them in sterile semen-saline solutions for 5 minutes. Swabs were kept frozen at −20°C until processed for DNA. To determine the efficacy and sensitivity of the assay, self-collected vaginal swabs (SCVS) were collected from female volunteers following unprotected intercourse with their partners and tested for the presence of Y-chromosomal DNA. SCVS from female volunteers abstaining from sexual intercourse were also collected and used as negative controls. Samples from our previous study2 were also evaluated using the newly developed assay, and the results compared with those previously obtained by the PCR and gel electrophoresis detection method.
Vaginal swabs were placed in 0.5 mL of TE buffer containing10 mmol/L Tris-HCl 7.5 and 10 mmol/L EDTA, incubated at room temperature for 25 minutes, and rotated vigorously to remove excess liquid. The sample was centrifuged for 3 minutes at 12,500 rpm and 100 μL of the supernatant removed. The sample was extracted for DNA using a multistep extraction protocol. First, 2 μL of proteinase-K solution (10 mg/mL) containing 2 mmol/L CaCl2, 10 mmol/mL Tris-HCl 7.5 in 40% glycerol was added to the specimen, gently inverted, and incubated for 30 minutes at 56°C. The specimen was then centrifuged for 3 minutes at 12,500 rpm and 100 μL of the supernatant removed. Two hundred microliters of a proteinase-K (10 mg/mL)/DTT (40 mmol) solution was added to the specimen, vortexed, and incubated in a sonicator bath at 56°C for 30 minutes. The sample was centrifuged as previously described and boiled for 10 minutes. Finally, 100 μL of 5% (w/v) Chelex 100 was added to the sample, incubated for 30 minutes at 56°C, and the supernatant used for polymerase chain reaction (PCR) amplification.
Amplification of Y Chromosomal DNA
The real-time PCR assay was carried out on the Roche LightCycler® v1.2 system (Roche Diagnostics, Indianapolis, IN). The instrument allows for the detection of PCR products by collecting fluorescence data from the accumulation of nucleic acid during the annealing step of each cycle. Detection of PCR amplification products is mediated by 2 fluorophore-labeled hybridization probes; 1 probe labeled at the 3′ end with fluorescein, and the second at the 5′ end with the LightCycler Red fluorophore. During the reaction, fluorescein, which serves as the donor molecule, is excited by the light source of the LightCycler, resulting in the transfer of resonance energy to the acceptor fluorophore molecule, emitting light of a higher wavelength resulting from fluorescence resonance energy transfer (FRET). The emitted florescence can be detected by the photohybrids of the instruments using 3 channels with different emission wavelengths (530, 640, and 705 nm), allowing for exact measurements of emission from the fluorophores. The amplification reactions take place in specially designed, sealed glass capillaries, thereby eliminating the possibility of PCR product contamination. The glass capillaries, and the air-driven temperature control of the instrument allows for rapid PCR. An entire 45-cycle run can be completed in approximately 45 minutes. In addition, PCR products can be easily characterized by melt curve analysis.
The amplification reactions were carried out in glass capillaries in 20 μL reactions containing 5 μL of extracted DNA, 4 μL of 1× LightCycler Fast Start Reaction Mix (Roche), 1 μmol/L of SRY-F 5′-ATTGGCGATTAAGTCAAATTCG-3′, and 0.5 μmol/L of SRY-R 5′-CCCCTAGTACCCTGACAATGT-3′ primers directly amplifying a 138 bp fragment of the SRY gene. The reactions also included 0.25 μmol/L of hybridization probes labeled with fluorescein (FL) or fluorophore molecules (Red 640):5′-CGTTGACTACTTGCCCTGCTGATCT-[FL]3′—and 5′ [Red 640]-C CTCCCTGACTGCTCTACTGCTGTC-3′—PH. Every run included a positive control as well as a negative control containing 5 μL of sterile PCR-grade water and reaction mix. The amplification parameters included an initial denaturation step at 95°C for 10 minutes, followed by 45 cycles of denaturation at 95°C for 10 seconds, primer and probe annealing at 53°C for 10 seconds and extension at 72°C for 7 seconds. The products were analyzed by melting curve analysis by applying 95°C at 0.1 second and 53°C at 5 seconds, followed by an increase in temperature from 53°C to 85°C (0.1°C/second) and continuous fluorescence recording.
Quantification of Amplified DNA
The amplified DNA was quantified by utilizing the absolute quantification analysis module of the LightCycler instrument, which calculates the concentration of target DNA in unknown samples, based on the concentration of standard samples. To quantify the DNA concentration of unknown samples, a standard curve was generated from a synthetic oligonucleotide (MWG Oligo, Ebersberg, Germany) corresponding to the 138 bp fragment of the SRY gene.
The real-time PCR assay can detect down to 10 copies of Y- chromosomal DNA (Fig. 1), which is comparable to our previous assay. The assay produced positives and reproducible results when tested with unknown experimental samples obtained at different time points after unprotected intercourse (Fig. 2a). The success of the reaction was determined by comparing the melting curve profile of the standard with that of the experimental unknown samples. All the experimental samples gave a distinct T m of 61.9°C (±0.25°C) identical to that of the standard control (Fig. 2b).
To further characterize the assay, a 2-step approach was undertaken. First, Yc-positive and -negative samples from the initial study2 were tested with the newly-developed assay. Second, SCVS collected every other day postcoital for a total of 14 days were analyzed for Y-chromosomal DNA. A typical graph, shown in Figure 3, demonstrates the decay of Y-PCR product signal over a 1-week period. Similar amplification curves were observed for all Yc-positive samples tested, and Yc DNA sequences were detected for several samples on days 9 and 11. The detection limit of the assay is similar to that of our previous study, which indicated complete disappearance of the Y-chromosomal signal by day 14. The assay correctly identified all Yc-positive and -negative samples from the initial study (data not shown). Additionally, Yc concentrations for all positive samples were comparable between the gel electrophoresis detection method and the newly developed real-time PCR assay.
Our goal was to develop a rapid, quantitative real-time PCR assay for the detection of Y-chromosomal (Yc) DNA by optimizing our previously reported assay.2 The DNA extraction protocol was optimized by using spiked SCVS with known concentrations of semen. The sensitivity of the assay was evaluated by using standards with known concentrations of DNA template.
Our RT-PCR assay detection limit of 10 copies of Yc DNA is comparable to our previous assay and is more time- and cost-effective. The previous assay required amplification of PCR products followed by visualization by gel electrophoresis, which required approximately 4 hours to complete. Amplification and detection of Yc sequences is conducted simultaneously by the LightCycler instrument allowing for detection and quantification of PCR products in approximately 45 minutes. The closed-capillary system also greatly reduces the potential for cross reactivity between samples. Another feature of the new assay is the potential for simultaneous melt curve analysis facilitating PCR product identification. Since the assay utilizes instruments that are increasingly available in diagnostic and public health laboratories, the Yc detection assay by real-time PCR approach can be more widely utilized by behavioral and clinical researchers.
1. Jadack RA, Yuenger J, Ghanem KG, Zenilman J. Polymerase chain reaction detection of Y-chromosome sequences in vaginal fluid of women accessing a sexually transmitted disease clinic. Sex Transm Dis 2006; 33:22–25.
© Copyright 2007 American Sexually Transmitted Diseases Association
2. Zenilman JM, Yuenger J, Galai N, Turner CF, Rogers SM. Polymerase chain reaction detection of Y chromosome sequences in vaginal fluid: Preliminary studies of a potential biomarker for sexual behavior. Sex Transm Dis 2005; 32:90–94.