Invader Plus Method Detects Herpes Simplex Virus in Cerebrospinal Flui
http://www.100md.com
《微生物临床杂志》
Third Wave Technologies, Inc., Madison, Wisconsin 53719
Vanderbilt University Medical Center, Nashville, Tennessee 37232-531
ABSTRACT
We report here on the development and validation of a prototype Invader Plus method for the qualitative detection of herpes simplex virus types 1 and 2 in cerebrospinal fluid (CSF). The method combines PCR and Invader techniques in a single, closed-tube, continuous-reaction format that gives an analytical sensitivity of approximately 10 copies per reaction. The clinical sensitivity and specificity were 100.0% and 98.6%, respectively, when the results of the method were validated against the results obtained with a PCR colorimetric microtiter plate system by use of clinical CSF specimens.
TEXT
Herpes simplex virus (HSV) type 1 (HSV-1) and HSV-2 are among the most widespread viruses known to cause a wide variety of acute and recurrent infections in humans. In addition to epithelial infections, such as gingivostomatitis, pharyngitis, genital herpes, whitlow, and keratoconjunctivitis, HSV is an important cause of central nervous system (CNS) infections, including necrotizing encephalitis and meningitis (10, 17, 24). Because specific antiviral therapy is available, a rapid, accurate, and definitive laboratory diagnosis of HSV infection is critical to support clinical findings.
Standard PCR-based methods are among the most widely used for the sensitive detection of HSV viral DNA in clinical specimens (3, 5, 6, 9, 13, 20, 23). The rapid and sensitive detection of HSV by real-time PCR has also been reported (4, 5, 7, 14, 22) with genotyping, and differentiation between HSV types 1 and 2 is possible through analysis of the melting curve of the PCR product (2). However, atypical melting curves that do not conform to the expected melting temperatures for HSV types 1 and 2 have been observed, and thus, an alternative genotyping assay must be used to resolve the results for such samples (2).
We present an alternative molecular method for the qualitative detection and differentiation of HSV-1 and HSV-2 by use of the Invader Plus method. Invader Plus combines PCR and Invader techniques in a single-tube, continuous-reaction format in which the detection of HSV and the differentiation between types 1 and 2 occur simultaneously. In this study a panel of clinical cerebrospinal fluid (CSF) specimens were used to validate the performance of this method by comparing its results to those obtained with a PCR colorimetric microtiter plate (PCR-enzyme immunoassay [EIA]) system, which has been routinely used for the clinical diagnosis of HSV infection (17, 21). The prototype method is sensitive, with a detection limit of approximately 10 copies per reaction. The assay is specific, combining the specificities of both the PCR and the Invader technologies, and rapid, with less than a 2-h reaction time; and since the signal readout is configured for end-point fluorescence detection, expensive instrumentation is not required.
Sample acquisition and extraction. HSV-1 and HSV-2 control strains with known titers were purchased from Advanced Biotechnologies Inc. (Columbia, MD). Viral particles were diluted to working levels in HSV-negative CSF (PCR negative) and extracted by use of a QIAamp DNA mini kit (QIAGEN Inc., Valencia, CA) by following the manufacturer's recommended procedure. To ensure extraction recovery and inhibition detection, an internal control (IC) standard supplied with the HSV reagents was spiked into the samples before the start of the extraction procedure by adding 5 μl of IC to the lysis buffer. The final elution volume of the extracted nucleic acids was 50 μl. For analytical sensitivity experiments, serial dilutions of HSV-1 and HSV-2 viral DNA (Advanced Biotechnologies Inc.) quantified by real-time PCR (18, 19) were made in a solution containing 20 ng/μl tRNA (Sigma-Aldrich, St. Louis, MO). For analytical specificity experiments, viral nucleic acids extracted from varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, human herpesvirus 6, Haemophilus influenzae, Neisseria meningitidis, Cryptococcus neoformans, and hepatitis B virus were obtained from Advanced Biotechnologies Inc (Columbia, MD) and the American Type Culture Collection (Manassas, VA) and were tested at 10,000 copies per reaction mixture by using the HSV Invader Plus reagents. An additional 101 CSF specimens submitted to the Molecular Infectious Diseases Laboratory at the Vanderbilt University Medical Center for the diagnosis of HSV CNS disease were selected retrospectively for the study. Nucleic acid from 200 μl of each CSF specimen was extracted by using a QIAamp MinElute virus spin kit (QIAGEN Inc.), according to the manufacturer's instructions (16). The extracted DNAs were resuspended in 50 μl of water, and 15 μl of each specimen extract was used for amplification.
Invader Plus method. Invader Plus combines PCR and the Invader reaction (11, 12) in a single-tube format and therefore combines target amplification through PCR and signal amplification through Invader chemistry. The enzymes used in Invader Plus are native Taq polymerase (Promega Corporation, Madison, WI) and Cleavase enzyme (Third Wave Technologies, Madison, WI). A schematic of the principles of Invader Plus is shown in Fig. 1. The reaction is configured to use PCR primers (depicted in yellow in Fig. 1) with melting temperature (Tms) of 72°C and Invader detection probe (depicted in green in Fig. 1) with a target-specific Tm of 63°C. The melting temperatures are calculated by using DNA nearest-neighbor thermodynamics (1) with appropriate salt correction factors (15). One of the PCR primers overlaps the probe by one nucleotide, thus forming the overlap flap substrate for the Cleavase enzyme at 63°C (Fig. 1A). The reactions in this study were carried out in a 96-well reaction plate with a 50-μl reaction volume. Fifteen microliters of reaction buffer, 15 μl of HSV-1- and HSV-2-specific oligonucleotide mixture, and 5 μl of enzyme mix were preassembled in a clean reagents hood and then transferred to target hood, where 15 μl of the target was added. The first step of Invader Plus is PCR target amplification, in which the reaction is subjected to 25 to 35 cycles of a denaturation step (95°C for 15 s) and hybridization and extension steps (72°C for 45 s) (Fig. 1B). At the end of PCR cycling, the reaction mixture is incubated at 99°C for 10 min to inactivate the Taq polymerase. Next, the reaction temperature is lowered to 63°C for 15 to 30 min to permit the hybridization of the probe oligonucleotide and the formation of an overlap flap structure (Fig. 1C). Upon the formation of the overlap flap structure, the Cleavase enzyme cleaves the 5' flap (colored in magenta in Fig. 1) downstream of the first base pair between the probe and the target. This releases a single-stranded 5' flap oligonucleotide that is then used for signal amplification in a secondary reaction detection system. In the secondary reaction, the cleaved flap hybridizes to a fluorescence resonance energy transfer (FRET) hairpin oligonucleotide (FRET cassette; depicted in orange in Fig. 1) containing a dye-quencher pair (F and Q, respectively) to form a secondary substrate for Cleavase enzyme. Once this substrate is formed, the 5' fluorophore-containing base (F) is cleaved, thus generating a fluorescence signal. The Invader reaction is performed at the Tms of both the probe and the cleaved flap to allow multiple cleavage events per target and cleaved flap (12). Data were then collected by using a GENios FL plate reader (Tecan, Durham, NC) equipped with 6-carboxyfluorescein (FAM) for HSV-1 detection (excitation and emission wavelengths, 485 nm and 520 nm, respectively), Redmond Red for HSV-2 detection (excitation and emission wavelengths, 578 nm and 650 nm, respectively), and Yakima Yellow for IC detection (excitation and emission wavelengths, 532 and 568 nm, respectively).
HSV Invader Plus reagents. The Invader Plus reagents are multiplexed for the simultaneous detection of several targets by using different target-specific primers and Invader reaction signal probes. These probes have 5' flaps that report to FRET cassettes with different dyes. The Invader Plus reagents are configured to incorporate dUTPs, thus allowing the user to use uracil-N-glycosylase (UNG) to minimize cross-contamination due to amplicon "carryover" from previous reactions (8). The HSV Invader Plus reagents and the Invader Plus core reagents (prototype reagents; Third Wave Technologies, Inc.) were used as directed by the manufacturer. Invader Plus primers and probe were designed to target the thymidine kinase genes of both HSV-1 and HSV-2.
Invader Plus data analysis. Raw data from the plate reader were exported and analyzed by using Microsoft Excel 2000 software. Fold-over-zero (FOZ) values were calculated for standards and unknown samples by dividing the raw signal obtained for each sample by the raw signal of the standard no-target control. FOZ values equal to or greater than 2.0 were considered positive signals. For unknown samples, a FOZ value of less than 2.0 for the IC and either HSV-1 or HSV-2 was considered an invalid result due to sample loss or the presence of inhibitors during sample extraction.
PCR-EIA. A PCR-EIA was performed to detect and differentiate HSV-1 and HSV-2 DNA by the procedure published previously (17, 21). Fifty-microliter PCR mixtures were set up to contain 1x buffer; 1.5 mM MgCl2; 10% glycerol; 200 μM dATP, dCTP, and dGTP; 100 μM dTTP; 90 μM dUTP; 10 μM digoxigenin-11-dUTP (Roche Biochemicals, Indianapolis, IN); 1 μM each primer; 0.01 units/μl UNG (Epicenter Technologies, Madison, WI); 0.025 units/μl AmpliTaq gold DNA polymerase (Applied Biosystems); and 5 μl of specimen DNA extract. The reaction mixtures were placed in an ABI 9700 thermal cycler (Applied Biosystems) programmed for a three-step PCR procedure, followed by an initial UNG activation, as described previously (21). The amplification products were identified by detecting digoxigenin-labeled PCR products with type-specific 5'-biotinylated capture probes for HSV-1 and HSV-2 (21). The output signal was measured at an optical density of 450 (OD450). A positive result was defined as a value of the OD450 – the OD490 greater than or equal to 0.1.
Level-of-detection experiments with eight replicates of samples with HSV-1 and HSV-2 viral DNA combined at levels ranging from 160 to 10 copies per reaction mixture resulted in the detection of seven of eight replicates for the 10 copies of HSV-1 per reaction and eight of eight replicates for the 10 copies of HSV-2 (data not shown). Greater than 10 copies per reaction mixture resulted in 100% detection for all replicates for both HSV-1 and HSV-2. The reaction is configured to be qualitative in nature, reporting the presence or the absence of either HSV-1 or HSV-2, or both. As a result, the FOZ values for both HSV-1 (reporting to FAM) and HSV-2 (reporting to Redmond Red) are 10 and 20, respectively, regardless of the target levels. (The FOZ values for HSV-2 are higher than those for HSV-1, presumably due to better filtering of the emission noise in the Redmond Red dye filters than in the FAM dye filters.)
To assess the specificity of the HSV Invader Plus reagents, purified nucleic acids from eight species (see above) were used. DNA target levels of 10,000 copies of each species were tested in triplicate, and for the FAM and Redmond Red dyes (HSV-1 and HSV-2 signals, respectively), FOZ values of 2.0 were obtained only when HSV-1 (FAM) or HSV-2 (Redmond Red) was present (data not shown). This indicates that the Invader Plus reagents for HSV-1 and HSV-2 detection are specific, in that they distinguish between types 1 and 2 of HSV and do not detect DNAs other than those of HSV in specimens.
To validate the performance of the HSV Invader Plus reagents, the PCR-EIA method (21) was used to compare the results obtained by using the same samples. For this study, 101 clinical CSF samples were extracted and tested by using the HSV Invader Plus reagents and PCR-EIA methods. The results are listed in Table 1. For HSV-1 detection, both methods detected 12 positive samples. For HSV-2 detection, the Invader Plus reagents detected 20 positive samples, while the PCR-EIA detected 19 positive samples. The discordant specimen was reextracted and retested, and the same results were obtained. Therefore, by using the results generated by the PCR-EIA method as a reference, the sensitivity and specificity of the HSV Invader Plus reagents were 100.0% and 98.6%, respectively. Finally, a 96.9% agreement was reached between the two assays, Invader Plus and PCR-EIA, for the differentiation of HSV types 1 and 2.
In this study, we report on the development of the Invader Plus method for the qualitative detection of HSV types 1 and 2 and its application for the sensitive and specific detection of HSV-1 and HSV-2 in CSF. The Invader Plus method combines the powers of target amplification of PCR and signal amplification of Invader chemistry (12). The combination of both amplification methods allows the ultrasensitive detection of DNA targets without compromising specificity. In the Invader Plus reaction, the specificities of both PCR and the Invader reaction are combined, therefore increasing the specificity obtained by running either method alone. The reaction is currently configured to be qualitative, but it can easily be configured to become quantitative by lowering the number of PCR cycles and slightly modifying the Invader reagents components (data not shown). The HSV Invader Plus method is capable of detecting 10 copies of either virus per reaction. Furthermore, we demonstrated the high specificity of the assay by showing the absence of cross-reactivity by using HSV reagents that interrogated samples containing 10,000 copies of the DNA of eight different species that are commonly found in CSF samples.
A direct comparison of the Invader Plus and the PCR-EIA methods for HSV detection with 101 blinded CSF specimens showed remarkable agreement between the two methods (Table 1). A single discordant result was obtained for a sample that was typed as HSV-2 positive by the Invader Plus method and negative by PCR-EIA. Retesting with a new extraction of the same sample resulted in similar calls. A possible explanation of the results obtained for the discordant sample is the presence of low numbers of copies of HSV-2, which made it possible for the Invader Plus method but not the PCR-EIA method to detect the HSV-2 DNA. Unfortunately, we are unable to confirm this explanation at this point since that sample was consumed in the study, thus preventing us from further investigating the sample using an alternative HSV detection method.
The Invader Plus detection method is configured for end-point fluorescence detection readout. The fluorescent reporter dyes used in the HSV detection reaction are compatible with common fluorescence microtiter plate readers equipped with appropriate FAM, Redmond Red, and Yakima Yellow excitation and emission filters (see above). Examples of the plate readers that were used for Invader Plus detection include CytoFluor 4000 (Applied Biosystems), GENios FL and Safire (Tecan), and FLx800 (BioTek Instruments). Real-time PCR instruments with end-point fluorescence readout capabilities, such as ABI 7500 (Applied Biosystems), Rotor-Gene 3000 (Corbett Life Science), and Chromo 4 (MJ Research), are also compatible with Invader Plus detection.
In summary, the Invader Plus method described in this study is a highly sensitive and specific method that is performed in a single closed-tube reaction to detect and distinguish between both types of HSV. The reaction is configured for end-point fluorescence detection and is compatible with most fluorescence plate readers. The reaction time is less than 2 h, which allows the method to be a rapid and relevant diagnostic procedure for the detection of HSV in CSF.
ACKNOWLEDGMENTS
We thank Steve Day, Kwok Wu, Jeff Hall, Susan Sefers, Shufang Meng, Thomas Smalling, Rebacah Glisson, Sharon Smith, and Mary Beth Underwood for their expert advice and technical assistance.
Invader, Cleavase, and Invader Plus are trademarks of Third Wave Technologies, Inc.
FOOTNOTES
Corresponding author. Mailing address: Third Wave Technologies, Inc., 502 South Rosa Rd., Madison, WI 53719. Phone: (608) 273-8933, ext. 8203. Fax: (608) 273-8618. E-mail: hallawi@twt.com.
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Vanderbilt University Medical Center, Nashville, Tennessee 37232-531
ABSTRACT
We report here on the development and validation of a prototype Invader Plus method for the qualitative detection of herpes simplex virus types 1 and 2 in cerebrospinal fluid (CSF). The method combines PCR and Invader techniques in a single, closed-tube, continuous-reaction format that gives an analytical sensitivity of approximately 10 copies per reaction. The clinical sensitivity and specificity were 100.0% and 98.6%, respectively, when the results of the method were validated against the results obtained with a PCR colorimetric microtiter plate system by use of clinical CSF specimens.
TEXT
Herpes simplex virus (HSV) type 1 (HSV-1) and HSV-2 are among the most widespread viruses known to cause a wide variety of acute and recurrent infections in humans. In addition to epithelial infections, such as gingivostomatitis, pharyngitis, genital herpes, whitlow, and keratoconjunctivitis, HSV is an important cause of central nervous system (CNS) infections, including necrotizing encephalitis and meningitis (10, 17, 24). Because specific antiviral therapy is available, a rapid, accurate, and definitive laboratory diagnosis of HSV infection is critical to support clinical findings.
Standard PCR-based methods are among the most widely used for the sensitive detection of HSV viral DNA in clinical specimens (3, 5, 6, 9, 13, 20, 23). The rapid and sensitive detection of HSV by real-time PCR has also been reported (4, 5, 7, 14, 22) with genotyping, and differentiation between HSV types 1 and 2 is possible through analysis of the melting curve of the PCR product (2). However, atypical melting curves that do not conform to the expected melting temperatures for HSV types 1 and 2 have been observed, and thus, an alternative genotyping assay must be used to resolve the results for such samples (2).
We present an alternative molecular method for the qualitative detection and differentiation of HSV-1 and HSV-2 by use of the Invader Plus method. Invader Plus combines PCR and Invader techniques in a single-tube, continuous-reaction format in which the detection of HSV and the differentiation between types 1 and 2 occur simultaneously. In this study a panel of clinical cerebrospinal fluid (CSF) specimens were used to validate the performance of this method by comparing its results to those obtained with a PCR colorimetric microtiter plate (PCR-enzyme immunoassay [EIA]) system, which has been routinely used for the clinical diagnosis of HSV infection (17, 21). The prototype method is sensitive, with a detection limit of approximately 10 copies per reaction. The assay is specific, combining the specificities of both the PCR and the Invader technologies, and rapid, with less than a 2-h reaction time; and since the signal readout is configured for end-point fluorescence detection, expensive instrumentation is not required.
Sample acquisition and extraction. HSV-1 and HSV-2 control strains with known titers were purchased from Advanced Biotechnologies Inc. (Columbia, MD). Viral particles were diluted to working levels in HSV-negative CSF (PCR negative) and extracted by use of a QIAamp DNA mini kit (QIAGEN Inc., Valencia, CA) by following the manufacturer's recommended procedure. To ensure extraction recovery and inhibition detection, an internal control (IC) standard supplied with the HSV reagents was spiked into the samples before the start of the extraction procedure by adding 5 μl of IC to the lysis buffer. The final elution volume of the extracted nucleic acids was 50 μl. For analytical sensitivity experiments, serial dilutions of HSV-1 and HSV-2 viral DNA (Advanced Biotechnologies Inc.) quantified by real-time PCR (18, 19) were made in a solution containing 20 ng/μl tRNA (Sigma-Aldrich, St. Louis, MO). For analytical specificity experiments, viral nucleic acids extracted from varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, human herpesvirus 6, Haemophilus influenzae, Neisseria meningitidis, Cryptococcus neoformans, and hepatitis B virus were obtained from Advanced Biotechnologies Inc (Columbia, MD) and the American Type Culture Collection (Manassas, VA) and were tested at 10,000 copies per reaction mixture by using the HSV Invader Plus reagents. An additional 101 CSF specimens submitted to the Molecular Infectious Diseases Laboratory at the Vanderbilt University Medical Center for the diagnosis of HSV CNS disease were selected retrospectively for the study. Nucleic acid from 200 μl of each CSF specimen was extracted by using a QIAamp MinElute virus spin kit (QIAGEN Inc.), according to the manufacturer's instructions (16). The extracted DNAs were resuspended in 50 μl of water, and 15 μl of each specimen extract was used for amplification.
Invader Plus method. Invader Plus combines PCR and the Invader reaction (11, 12) in a single-tube format and therefore combines target amplification through PCR and signal amplification through Invader chemistry. The enzymes used in Invader Plus are native Taq polymerase (Promega Corporation, Madison, WI) and Cleavase enzyme (Third Wave Technologies, Madison, WI). A schematic of the principles of Invader Plus is shown in Fig. 1. The reaction is configured to use PCR primers (depicted in yellow in Fig. 1) with melting temperature (Tms) of 72°C and Invader detection probe (depicted in green in Fig. 1) with a target-specific Tm of 63°C. The melting temperatures are calculated by using DNA nearest-neighbor thermodynamics (1) with appropriate salt correction factors (15). One of the PCR primers overlaps the probe by one nucleotide, thus forming the overlap flap substrate for the Cleavase enzyme at 63°C (Fig. 1A). The reactions in this study were carried out in a 96-well reaction plate with a 50-μl reaction volume. Fifteen microliters of reaction buffer, 15 μl of HSV-1- and HSV-2-specific oligonucleotide mixture, and 5 μl of enzyme mix were preassembled in a clean reagents hood and then transferred to target hood, where 15 μl of the target was added. The first step of Invader Plus is PCR target amplification, in which the reaction is subjected to 25 to 35 cycles of a denaturation step (95°C for 15 s) and hybridization and extension steps (72°C for 45 s) (Fig. 1B). At the end of PCR cycling, the reaction mixture is incubated at 99°C for 10 min to inactivate the Taq polymerase. Next, the reaction temperature is lowered to 63°C for 15 to 30 min to permit the hybridization of the probe oligonucleotide and the formation of an overlap flap structure (Fig. 1C). Upon the formation of the overlap flap structure, the Cleavase enzyme cleaves the 5' flap (colored in magenta in Fig. 1) downstream of the first base pair between the probe and the target. This releases a single-stranded 5' flap oligonucleotide that is then used for signal amplification in a secondary reaction detection system. In the secondary reaction, the cleaved flap hybridizes to a fluorescence resonance energy transfer (FRET) hairpin oligonucleotide (FRET cassette; depicted in orange in Fig. 1) containing a dye-quencher pair (F and Q, respectively) to form a secondary substrate for Cleavase enzyme. Once this substrate is formed, the 5' fluorophore-containing base (F) is cleaved, thus generating a fluorescence signal. The Invader reaction is performed at the Tms of both the probe and the cleaved flap to allow multiple cleavage events per target and cleaved flap (12). Data were then collected by using a GENios FL plate reader (Tecan, Durham, NC) equipped with 6-carboxyfluorescein (FAM) for HSV-1 detection (excitation and emission wavelengths, 485 nm and 520 nm, respectively), Redmond Red for HSV-2 detection (excitation and emission wavelengths, 578 nm and 650 nm, respectively), and Yakima Yellow for IC detection (excitation and emission wavelengths, 532 and 568 nm, respectively).
HSV Invader Plus reagents. The Invader Plus reagents are multiplexed for the simultaneous detection of several targets by using different target-specific primers and Invader reaction signal probes. These probes have 5' flaps that report to FRET cassettes with different dyes. The Invader Plus reagents are configured to incorporate dUTPs, thus allowing the user to use uracil-N-glycosylase (UNG) to minimize cross-contamination due to amplicon "carryover" from previous reactions (8). The HSV Invader Plus reagents and the Invader Plus core reagents (prototype reagents; Third Wave Technologies, Inc.) were used as directed by the manufacturer. Invader Plus primers and probe were designed to target the thymidine kinase genes of both HSV-1 and HSV-2.
Invader Plus data analysis. Raw data from the plate reader were exported and analyzed by using Microsoft Excel 2000 software. Fold-over-zero (FOZ) values were calculated for standards and unknown samples by dividing the raw signal obtained for each sample by the raw signal of the standard no-target control. FOZ values equal to or greater than 2.0 were considered positive signals. For unknown samples, a FOZ value of less than 2.0 for the IC and either HSV-1 or HSV-2 was considered an invalid result due to sample loss or the presence of inhibitors during sample extraction.
PCR-EIA. A PCR-EIA was performed to detect and differentiate HSV-1 and HSV-2 DNA by the procedure published previously (17, 21). Fifty-microliter PCR mixtures were set up to contain 1x buffer; 1.5 mM MgCl2; 10% glycerol; 200 μM dATP, dCTP, and dGTP; 100 μM dTTP; 90 μM dUTP; 10 μM digoxigenin-11-dUTP (Roche Biochemicals, Indianapolis, IN); 1 μM each primer; 0.01 units/μl UNG (Epicenter Technologies, Madison, WI); 0.025 units/μl AmpliTaq gold DNA polymerase (Applied Biosystems); and 5 μl of specimen DNA extract. The reaction mixtures were placed in an ABI 9700 thermal cycler (Applied Biosystems) programmed for a three-step PCR procedure, followed by an initial UNG activation, as described previously (21). The amplification products were identified by detecting digoxigenin-labeled PCR products with type-specific 5'-biotinylated capture probes for HSV-1 and HSV-2 (21). The output signal was measured at an optical density of 450 (OD450). A positive result was defined as a value of the OD450 – the OD490 greater than or equal to 0.1.
Level-of-detection experiments with eight replicates of samples with HSV-1 and HSV-2 viral DNA combined at levels ranging from 160 to 10 copies per reaction mixture resulted in the detection of seven of eight replicates for the 10 copies of HSV-1 per reaction and eight of eight replicates for the 10 copies of HSV-2 (data not shown). Greater than 10 copies per reaction mixture resulted in 100% detection for all replicates for both HSV-1 and HSV-2. The reaction is configured to be qualitative in nature, reporting the presence or the absence of either HSV-1 or HSV-2, or both. As a result, the FOZ values for both HSV-1 (reporting to FAM) and HSV-2 (reporting to Redmond Red) are 10 and 20, respectively, regardless of the target levels. (The FOZ values for HSV-2 are higher than those for HSV-1, presumably due to better filtering of the emission noise in the Redmond Red dye filters than in the FAM dye filters.)
To assess the specificity of the HSV Invader Plus reagents, purified nucleic acids from eight species (see above) were used. DNA target levels of 10,000 copies of each species were tested in triplicate, and for the FAM and Redmond Red dyes (HSV-1 and HSV-2 signals, respectively), FOZ values of 2.0 were obtained only when HSV-1 (FAM) or HSV-2 (Redmond Red) was present (data not shown). This indicates that the Invader Plus reagents for HSV-1 and HSV-2 detection are specific, in that they distinguish between types 1 and 2 of HSV and do not detect DNAs other than those of HSV in specimens.
To validate the performance of the HSV Invader Plus reagents, the PCR-EIA method (21) was used to compare the results obtained by using the same samples. For this study, 101 clinical CSF samples were extracted and tested by using the HSV Invader Plus reagents and PCR-EIA methods. The results are listed in Table 1. For HSV-1 detection, both methods detected 12 positive samples. For HSV-2 detection, the Invader Plus reagents detected 20 positive samples, while the PCR-EIA detected 19 positive samples. The discordant specimen was reextracted and retested, and the same results were obtained. Therefore, by using the results generated by the PCR-EIA method as a reference, the sensitivity and specificity of the HSV Invader Plus reagents were 100.0% and 98.6%, respectively. Finally, a 96.9% agreement was reached between the two assays, Invader Plus and PCR-EIA, for the differentiation of HSV types 1 and 2.
In this study, we report on the development of the Invader Plus method for the qualitative detection of HSV types 1 and 2 and its application for the sensitive and specific detection of HSV-1 and HSV-2 in CSF. The Invader Plus method combines the powers of target amplification of PCR and signal amplification of Invader chemistry (12). The combination of both amplification methods allows the ultrasensitive detection of DNA targets without compromising specificity. In the Invader Plus reaction, the specificities of both PCR and the Invader reaction are combined, therefore increasing the specificity obtained by running either method alone. The reaction is currently configured to be qualitative, but it can easily be configured to become quantitative by lowering the number of PCR cycles and slightly modifying the Invader reagents components (data not shown). The HSV Invader Plus method is capable of detecting 10 copies of either virus per reaction. Furthermore, we demonstrated the high specificity of the assay by showing the absence of cross-reactivity by using HSV reagents that interrogated samples containing 10,000 copies of the DNA of eight different species that are commonly found in CSF samples.
A direct comparison of the Invader Plus and the PCR-EIA methods for HSV detection with 101 blinded CSF specimens showed remarkable agreement between the two methods (Table 1). A single discordant result was obtained for a sample that was typed as HSV-2 positive by the Invader Plus method and negative by PCR-EIA. Retesting with a new extraction of the same sample resulted in similar calls. A possible explanation of the results obtained for the discordant sample is the presence of low numbers of copies of HSV-2, which made it possible for the Invader Plus method but not the PCR-EIA method to detect the HSV-2 DNA. Unfortunately, we are unable to confirm this explanation at this point since that sample was consumed in the study, thus preventing us from further investigating the sample using an alternative HSV detection method.
The Invader Plus detection method is configured for end-point fluorescence detection readout. The fluorescent reporter dyes used in the HSV detection reaction are compatible with common fluorescence microtiter plate readers equipped with appropriate FAM, Redmond Red, and Yakima Yellow excitation and emission filters (see above). Examples of the plate readers that were used for Invader Plus detection include CytoFluor 4000 (Applied Biosystems), GENios FL and Safire (Tecan), and FLx800 (BioTek Instruments). Real-time PCR instruments with end-point fluorescence readout capabilities, such as ABI 7500 (Applied Biosystems), Rotor-Gene 3000 (Corbett Life Science), and Chromo 4 (MJ Research), are also compatible with Invader Plus detection.
In summary, the Invader Plus method described in this study is a highly sensitive and specific method that is performed in a single closed-tube reaction to detect and distinguish between both types of HSV. The reaction is configured for end-point fluorescence detection and is compatible with most fluorescence plate readers. The reaction time is less than 2 h, which allows the method to be a rapid and relevant diagnostic procedure for the detection of HSV in CSF.
ACKNOWLEDGMENTS
We thank Steve Day, Kwok Wu, Jeff Hall, Susan Sefers, Shufang Meng, Thomas Smalling, Rebacah Glisson, Sharon Smith, and Mary Beth Underwood for their expert advice and technical assistance.
Invader, Cleavase, and Invader Plus are trademarks of Third Wave Technologies, Inc.
FOOTNOTES
Corresponding author. Mailing address: Third Wave Technologies, Inc., 502 South Rosa Rd., Madison, WI 53719. Phone: (608) 273-8933, ext. 8203. Fax: (608) 273-8618. E-mail: hallawi@twt.com.
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