FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1977436377> ?p ?o ?g. }

• W1977436377 endingPage "619" @default.
• W1977436377 startingPage "617" @default.
• W1977436377 abstract "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furthermore, condoms were effective in preventing transmission of detectable Y-chromosome material to women (Ghanem KG-Unpublished data—submitted as companion publication).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. Sample Collection 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. DNA Extraction 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 Tm of 61.9°C (±0.25°C) identical to that of the standard control (Fig. 2b).Fig. 1: Sensitivity of the real-time PCR assay with purified PCR product from a synthetic DNA template corresponding to a 138 bp of the SRY gene (6 × 105 to 1 × 101 copies).Fig. 2: (a) Decay of the Y-chromosomal DNA signal over a 24-hour period. A series of self-administered vaginal swabs were collected as follows: A: Postcoital, B: 12 hours postcoital, and C: 24 hours postcoital, and tested for the presence of Y-chromosomal DNA. A sample collected before intercourse served as negative control. (b) Melting curve analysis of the experimental samples A, B, C, and standard control D. All experimental samples display identical melt curve as the standard control. For simplicity, the standard control (D) was omitted from Figure 2a.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.Fig. 3: Decay of the Y-chromosomal DNA signal over a 1-week period following unprotected intercourse. The negative control was obtained before intercourse.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." @default.
• W1977436377 created "2016-06-24" @default.
• W1977436377 creator A5006602630 @default.
• W1977436377 creator A5011188586 @default.
• W1977436377 creator A5017779608 @default.
• W1977436377 creator A5019470982 @default.
• W1977436377 creator A5045328465 @default.
• W1977436377 creator A5056437210 @default.
• W1977436377 creator A5060818509 @default.
• W1977436377 date "2007-08-01" @default.
• W1977436377 modified "2023-09-27" @default.
• W1977436377 title "Detection and Quantification of Y-Chromosomal Sequences by Real-Time PCR Using the LightCycler® System" @default.
• W1977436377 cites W1966137936 @default.
• W1977436377 cites W2137500297 @default.
• W1977436377 doi "https://doi.org/10.1097/01.olq.0000258336.65285.31" @default.
• W1977436377 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17334265" @default.
• W1977436377 hasPublicationYear "2007" @default.
• W1977436377 type Work @default.
• W1977436377 sameAs 1977436377 @default.
• W1977436377 citedByCount "17" @default.
• W1977436377 countsByYear W19774363772013 @default.
• W1977436377 countsByYear W19774363772014 @default.
• W1977436377 countsByYear W19774363772016 @default.
• W1977436377 crossrefType "journal-article" @default.
• W1977436377 hasAuthorship W1977436377A5006602630 @default.
• W1977436377 hasAuthorship W1977436377A5011188586 @default.
• W1977436377 hasAuthorship W1977436377A5017779608 @default.
• W1977436377 hasAuthorship W1977436377A5019470982 @default.
• W1977436377 hasAuthorship W1977436377A5045328465 @default.
• W1977436377 hasAuthorship W1977436377A5056437210 @default.
• W1977436377 hasAuthorship W1977436377A5060818509 @default.
• W1977436377 hasBestOaLocation W19774363771 @default.
• W1977436377 hasConcept C104317684 @default.
• W1977436377 hasConcept C48023723 @default.
• W1977436377 hasConcept C54355233 @default.
• W1977436377 hasConcept C70721500 @default.
• W1977436377 hasConcept C71924100 @default.
• W1977436377 hasConcept C86803240 @default.
• W1977436377 hasConceptScore W1977436377C104317684 @default.
• W1977436377 hasConceptScore W1977436377C48023723 @default.
• W1977436377 hasConceptScore W1977436377C54355233 @default.
• W1977436377 hasConceptScore W1977436377C70721500 @default.
• W1977436377 hasConceptScore W1977436377C71924100 @default.
• W1977436377 hasConceptScore W1977436377C86803240 @default.
• W1977436377 hasIssue "8" @default.
• W1977436377 hasLocation W19774363771 @default.
• W1977436377 hasLocation W19774363772 @default.
• W1977436377 hasLocation W19774363773 @default.
• W1977436377 hasOpenAccess W1977436377 @default.
• W1977436377 hasPrimaryLocation W19774363771 @default.
• W1977436377 hasRelatedWork W1535303881 @default.
• W1977436377 hasRelatedWork W19166555 @default.
• W1977436377 hasRelatedWork W1971602763 @default.
• W1977436377 hasRelatedWork W2041332439 @default.
• W1977436377 hasRelatedWork W2089145757 @default.
• W1977436377 hasRelatedWork W2110784088 @default.
• W1977436377 hasRelatedWork W2128088040 @default.
• W1977436377 hasRelatedWork W2168712887 @default.
• W1977436377 hasRelatedWork W2363233029 @default.
• W1977436377 hasRelatedWork W2385826369 @default.
• W1977436377 hasVolume "34" @default.
• W1977436377 isParatext "false" @default.
• W1977436377 isRetracted "false" @default.
• W1977436377 magId "1977436377" @default.
• W1977436377 workType "article" @default.