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Wanchai Maleewong - One of the best experts on this subject based on the ideXlab platform.
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Molecular evidence of Opisthorchis viverrini in infected bithyniid snails in the Lao People's Democratic Republic by specific Hybridization Probe-based real-time fluorescence resonance energy transfer PCR method.
Parasitology Research, 2011Co-Authors: Pusadee Sri-aroon, Pewpan M Intapan, Chantima Lohachit, Phunthira Phongsasakulchoti, Tongjit Thanchomnang, Viraphong Lulitanond, Alexandra Hiscox, Samlane Phompida, Pany Sananikhom, Wanchai MaleewongAbstract:Naturally occurring bithyniid snails, Bithynia siamensis goniomphalos (Prosobranchia: Bithyniidae), and their intermediate hosts were sampled from Khammouane Province, the Lao People's Democratic Republic, and the prevalence of the carcinogenic human liver fluke, Opisthorchis viverrini, was examined. The presence of O. viverrini cercariae in snails was examined by cercarial shedding test and then confirmed by specific Hybridization Probe-based real-time fluorescence resonance energy transfer (FRET) PCR method. The real-time FRET PCR method is based on a fluorescence melting curve analysis of a hybrid between an amplicon produced from the pOV-A6 specific sequence (Genbank accession no. S80278), a 162-bp repeated sequence specific to O. viverrini, and specific fluorophore-labeled Probes. Mean melting temperature of O. viverrini DNA from the cercariae and each of two positive snails by shedding test was 66.3 ± 0.1. The O. viverrini infection rate in snails was 2.47% (2/81) by cercarial shedding test but was 8.52% (4/47) by real-time FRET PCR method. The real-time FRET PCR method is rapid and effective in examining a large number of snail samples simultaneously. Validation using molecular evidence from this procedure provides another tool for surveying the prevalence of O. viverrini-infected snails in Southeast Asian countries.
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Molecular evidence of Opisthorchis viverrini in infected bithyniid snails in the Lao People’s Democratic Republic by specific Hybridization Probe-based real-time fluorescence resonance energy transfer PCR method
Parasitology Research, 2011Co-Authors: Pusadee Sri-aroon, Pewpan M Intapan, Chantima Lohachit, Phunthira Phongsasakulchoti, Tongjit Thanchomnang, Viraphong Lulitanond, Alexandra Hiscox, Samlane Phompida, Pany Sananikhom, Wanchai MaleewongAbstract:Naturally occurring bithyniid snails, Bithynia siamensis goniomphalos (Prosobranchia: Bithyniidae), and their intermediate hosts were sampled from Khammouane Province, the Lao People's Democratic Republic, and the prevalence of the carcinogenic human liver fluke, Opisthorchis viverrini , was examined. The presence of O. viverrini cercariae in snails was examined by cercarial shedding test and then confirmed by specific Hybridization Probe-based real-time fluorescence resonance energy transfer (FRET) PCR method. The real-time FRET PCR method is based on a fluorescence melting curve analysis of a hybrid between an amplicon produced from the pOV-A6 specific sequence (Genbank accession no. S80278), a 162-bp repeated sequence specific to O. viverrini , and specific fluorophore-labeled Probes. Mean melting temperature of O. viverrini DNA from the cercariae and each of two positive snails by shedding test was 66.3 ± 0.1. The O. viverrini infection rate in snails was 2.47% (2/81) by cercarial shedding test but was 8.52% (4/47) by real-time FRET PCR method. The real-time FRET PCR method is rapid and effective in examining a large number of snail samples simultaneously. Validation using molecular evidence from this procedure provides another tool for surveying the prevalence of O. viverrini -infected snails in Southeast Asian countries.
Francisco Figueroa Roberto - One of the best experts on this subject based on the ideXlab platform.
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real time pcr detection of brucella abortus a comparative study of sybr green i 5 exonuclease and Hybridization Probe assays
Applied and Environmental Microbiology, 2003Co-Authors: Deborah Trishelle Newby, Ted L. Hadfield, Francisco Figueroa RobertoAbstract:Members of the bacterial genus Brucella infect a wide range of animal hosts, including cows, goats, sheep, pigs, dogs, elk, bison, and even marine mammals. The genus Brucella comprises six species: B. abortus, B. melitensis, B. suis, B. ovis, B. canis, and B. neotomae. It has been suggested from 16S rRNA gene sequence analysis and other biochemical characteristics (18, 29, 30) that the Brucella spp. constitute a monophyletic genus. Verger et al. (29, 30) proposed that the Brucella species be described as biovars of a single species, B. melitensis. However, Corbel (7) suggested that the existing nomenclature be retained for clarity. The extant literature associates particular species and biovars with brucellosis in different animal hosts: B. abortus with cows (also bison and elk), B. suis with pigs, B. ovis with sheep, B. melitensis with goats, and B. neotomae with desert wood rats. Brucellosis in bovine species can be associated specifically with B. abortus biovar 1, which accounts for 85% of reported cases (19). However, the ability of several Brucella species to infect organisms outside of their normal host range (e.g., B. melitensis and B. suis may infect cows, and B. melitensis, B. suis, B. abortus, and B. canis may infect humans) has been documented. This pathogen has historically had a major economic impact on U.S. agriculture, especially the dairy industry, where it is estimated that over $3.5 billion has been spent to eradicate bovine brucellosis. It also represents a risk to humans, not only through the consumption of infected meat or milk (2) but also through the deliberate deployment of the organism as a biological weapon. B. suis was the first microorganism developed in the former U.S. biological weapons program, primarily because of the ease with which it can be spread through aerosol routes of infection (12). The effects of brucellosis in humans (also known as undulant fever) are generally nonlethal, but can be extremely debilitating. Brucellosis remains a problem for U.S. agriculture today from the perspective of minimizing the potential for natural reservoirs of the disease to reinfect cattle, because brucellosis is nearly eradicated otherwise. As a consequence, wild bison and elk residing in areas such as Yellowstone National Park (YNP) are being studied to understand the relative risk wild animals pose to cattle ranging in the same region. This issue has generated intense interest after an Idaho cow was diagnosed with brucellosis in the spring of 2002. The long-term focus of the work reported here is to develop a rapid and sensitive real-time PCR assay for the specific detection of B. abortus in bison, cattle, and elk in the field. Diagnostic methods for brucellosis currently rely on serological tests that detect antibodies against Brucella and cultivation. Blood or tissue cultures remain the standard for diagnosis, but since these microorganisms can be spread by aerosol routes of exposure, controlling the risk of infection in laboratory workers requires the use of additional biological containment measures, such as biosafety cabinets. Significant effort has been expended to develop DNA diagnostics for brucellosis that exploit the selectivity and sensitivity of PCR. Gene targets have included outer membrane proteins (3, 4, 8, 11, 13, 16), 16S rRNA gene sequences (15), 16S-23S spacer regions (23), housekeeping genes (8), erythritol utilization genes (6), and insertion sequences (6, 20, 22). These assays have been applied to bacterial isolates, as well as clinical specimens, with limits of detection down to 10 to 100 fg of bacterial genomic DNA and cell numbers ranging from 10 to 7,000 cells per ml of blood (13, 16, 17) or milk (16, 17, 23, 24, 26). Single primer sets or multiplexed assays (5, 6, 10, 26) with a cocktail of primers permit the detection of most of the known species or biovars of Brucella by conventional PCR. A variety of PCR assays have been developed that can differentiate Brucella species; however, they typically rely on post-PCR analysis, such as amplicon size (5), size discrimination combined with restriction fragment length polymorphism analysis (4, 8), or randomly amplified polymorphic DNA analysis (27). Most recently, real-time PCR assays for the detection of Brucella spp. have been developed (22). Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation, measured by increased fluorescence during cycling. A number of different approaches can be used to generate the fluorescence signal. In this work, three approaches: SYBR Green I (a double-stranded DNA [dsDNA] intercalating dye), 5′-exonuclease (enzymatically released fluors), and Hybridization Probes (fluorescence resonance energy transfer [FRET]) were evaluated for real-time PCR detection of B. abortus. Although the 5′-exonuclease assay (TaqMan) is dependent upon the annealing of a single Probe to the target amplicon, in this article the term “Hybridization Probe assay” refers exclusively to the use of two adjacent Probes in which fluorescence is generated by resonance energy transfer from a donor fluor on one Probe to an acceptor fluor on the Probe 2 bp away.
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real time pcr detection of brucella abortus a comparative study of sybr green i 5 exonuclease and Hybridization Probe assays
Applied and Environmental Microbiology, 2003Co-Authors: Deborah Trishelle Newby, Ted L. Hadfield, Francisco Figueroa RobertoAbstract:Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence. A number of different approaches can be used to generate the fluorescence signal. Three approaches-SYBR Green I (a double-stranded DNA intercalating dye), 5'-exonuclease (enzymatically released fluors), and Hybridization Probes (fluorescence resonance energy transfer)-were evaluated for use in a real-time PCR assay to detect Brucella abortus. The three assays utilized the same amplification primers to produce an identical amplicon. This amplicon spans a region of the B. abortus genome that includes portions of the alkB gene and the IS711 insertion element. All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the Hybridization Probe assay.
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Real-Time PCR Detection of Brucella abortus: a Comparative Study of SYBR Green I, 5′-Exonuclease, and Hybridization Probe Assays
Applied and environmental microbiology, 2003Co-Authors: Deborah Trishelle Newby, Ted L. Hadfield, Francisco Figueroa RobertoAbstract:Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence. A number of different approaches can be used to generate the fluorescence signal. Three approaches-SYBR Green I (a double-stranded DNA intercalating dye), 5'-exonuclease (enzymatically released fluors), and Hybridization Probes (fluorescence resonance energy transfer)-were evaluated for use in a real-time PCR assay to detect Brucella abortus. The three assays utilized the same amplification primers to produce an identical amplicon. This amplicon spans a region of the B. abortus genome that includes portions of the alkB gene and the IS711 insertion element. All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the Hybridization Probe assay.
Deborah Trishelle Newby - One of the best experts on this subject based on the ideXlab platform.
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real time pcr detection of brucella abortus a comparative study of sybr green i 5 exonuclease and Hybridization Probe assays
Applied and Environmental Microbiology, 2003Co-Authors: Deborah Trishelle Newby, Ted L. Hadfield, Francisco Figueroa RobertoAbstract:Members of the bacterial genus Brucella infect a wide range of animal hosts, including cows, goats, sheep, pigs, dogs, elk, bison, and even marine mammals. The genus Brucella comprises six species: B. abortus, B. melitensis, B. suis, B. ovis, B. canis, and B. neotomae. It has been suggested from 16S rRNA gene sequence analysis and other biochemical characteristics (18, 29, 30) that the Brucella spp. constitute a monophyletic genus. Verger et al. (29, 30) proposed that the Brucella species be described as biovars of a single species, B. melitensis. However, Corbel (7) suggested that the existing nomenclature be retained for clarity. The extant literature associates particular species and biovars with brucellosis in different animal hosts: B. abortus with cows (also bison and elk), B. suis with pigs, B. ovis with sheep, B. melitensis with goats, and B. neotomae with desert wood rats. Brucellosis in bovine species can be associated specifically with B. abortus biovar 1, which accounts for 85% of reported cases (19). However, the ability of several Brucella species to infect organisms outside of their normal host range (e.g., B. melitensis and B. suis may infect cows, and B. melitensis, B. suis, B. abortus, and B. canis may infect humans) has been documented. This pathogen has historically had a major economic impact on U.S. agriculture, especially the dairy industry, where it is estimated that over $3.5 billion has been spent to eradicate bovine brucellosis. It also represents a risk to humans, not only through the consumption of infected meat or milk (2) but also through the deliberate deployment of the organism as a biological weapon. B. suis was the first microorganism developed in the former U.S. biological weapons program, primarily because of the ease with which it can be spread through aerosol routes of infection (12). The effects of brucellosis in humans (also known as undulant fever) are generally nonlethal, but can be extremely debilitating. Brucellosis remains a problem for U.S. agriculture today from the perspective of minimizing the potential for natural reservoirs of the disease to reinfect cattle, because brucellosis is nearly eradicated otherwise. As a consequence, wild bison and elk residing in areas such as Yellowstone National Park (YNP) are being studied to understand the relative risk wild animals pose to cattle ranging in the same region. This issue has generated intense interest after an Idaho cow was diagnosed with brucellosis in the spring of 2002. The long-term focus of the work reported here is to develop a rapid and sensitive real-time PCR assay for the specific detection of B. abortus in bison, cattle, and elk in the field. Diagnostic methods for brucellosis currently rely on serological tests that detect antibodies against Brucella and cultivation. Blood or tissue cultures remain the standard for diagnosis, but since these microorganisms can be spread by aerosol routes of exposure, controlling the risk of infection in laboratory workers requires the use of additional biological containment measures, such as biosafety cabinets. Significant effort has been expended to develop DNA diagnostics for brucellosis that exploit the selectivity and sensitivity of PCR. Gene targets have included outer membrane proteins (3, 4, 8, 11, 13, 16), 16S rRNA gene sequences (15), 16S-23S spacer regions (23), housekeeping genes (8), erythritol utilization genes (6), and insertion sequences (6, 20, 22). These assays have been applied to bacterial isolates, as well as clinical specimens, with limits of detection down to 10 to 100 fg of bacterial genomic DNA and cell numbers ranging from 10 to 7,000 cells per ml of blood (13, 16, 17) or milk (16, 17, 23, 24, 26). Single primer sets or multiplexed assays (5, 6, 10, 26) with a cocktail of primers permit the detection of most of the known species or biovars of Brucella by conventional PCR. A variety of PCR assays have been developed that can differentiate Brucella species; however, they typically rely on post-PCR analysis, such as amplicon size (5), size discrimination combined with restriction fragment length polymorphism analysis (4, 8), or randomly amplified polymorphic DNA analysis (27). Most recently, real-time PCR assays for the detection of Brucella spp. have been developed (22). Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation, measured by increased fluorescence during cycling. A number of different approaches can be used to generate the fluorescence signal. In this work, three approaches: SYBR Green I (a double-stranded DNA [dsDNA] intercalating dye), 5′-exonuclease (enzymatically released fluors), and Hybridization Probes (fluorescence resonance energy transfer [FRET]) were evaluated for real-time PCR detection of B. abortus. Although the 5′-exonuclease assay (TaqMan) is dependent upon the annealing of a single Probe to the target amplicon, in this article the term “Hybridization Probe assay” refers exclusively to the use of two adjacent Probes in which fluorescence is generated by resonance energy transfer from a donor fluor on one Probe to an acceptor fluor on the Probe 2 bp away.
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real time pcr detection of brucella abortus a comparative study of sybr green i 5 exonuclease and Hybridization Probe assays
Applied and Environmental Microbiology, 2003Co-Authors: Deborah Trishelle Newby, Ted L. Hadfield, Francisco Figueroa RobertoAbstract:Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence. A number of different approaches can be used to generate the fluorescence signal. Three approaches-SYBR Green I (a double-stranded DNA intercalating dye), 5'-exonuclease (enzymatically released fluors), and Hybridization Probes (fluorescence resonance energy transfer)-were evaluated for use in a real-time PCR assay to detect Brucella abortus. The three assays utilized the same amplification primers to produce an identical amplicon. This amplicon spans a region of the B. abortus genome that includes portions of the alkB gene and the IS711 insertion element. All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the Hybridization Probe assay.
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Real-Time PCR Detection of Brucella abortus: a Comparative Study of SYBR Green I, 5′-Exonuclease, and Hybridization Probe Assays
Applied and environmental microbiology, 2003Co-Authors: Deborah Trishelle Newby, Ted L. Hadfield, Francisco Figueroa RobertoAbstract:Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence. A number of different approaches can be used to generate the fluorescence signal. Three approaches-SYBR Green I (a double-stranded DNA intercalating dye), 5'-exonuclease (enzymatically released fluors), and Hybridization Probes (fluorescence resonance energy transfer)-were evaluated for use in a real-time PCR assay to detect Brucella abortus. The three assays utilized the same amplification primers to produce an identical amplicon. This amplicon spans a region of the B. abortus genome that includes portions of the alkB gene and the IS711 insertion element. All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the Hybridization Probe assay.
Alexandra Hiscox - One of the best experts on this subject based on the ideXlab platform.
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Molecular evidence of Opisthorchis viverrini in infected bithyniid snails in the Lao People's Democratic Republic by specific Hybridization Probe-based real-time fluorescence resonance energy transfer PCR method.
Parasitology Research, 2011Co-Authors: Pusadee Sri-aroon, Pewpan M Intapan, Chantima Lohachit, Phunthira Phongsasakulchoti, Tongjit Thanchomnang, Viraphong Lulitanond, Alexandra Hiscox, Samlane Phompida, Pany Sananikhom, Wanchai MaleewongAbstract:Naturally occurring bithyniid snails, Bithynia siamensis goniomphalos (Prosobranchia: Bithyniidae), and their intermediate hosts were sampled from Khammouane Province, the Lao People's Democratic Republic, and the prevalence of the carcinogenic human liver fluke, Opisthorchis viverrini, was examined. The presence of O. viverrini cercariae in snails was examined by cercarial shedding test and then confirmed by specific Hybridization Probe-based real-time fluorescence resonance energy transfer (FRET) PCR method. The real-time FRET PCR method is based on a fluorescence melting curve analysis of a hybrid between an amplicon produced from the pOV-A6 specific sequence (Genbank accession no. S80278), a 162-bp repeated sequence specific to O. viverrini, and specific fluorophore-labeled Probes. Mean melting temperature of O. viverrini DNA from the cercariae and each of two positive snails by shedding test was 66.3 ± 0.1. The O. viverrini infection rate in snails was 2.47% (2/81) by cercarial shedding test but was 8.52% (4/47) by real-time FRET PCR method. The real-time FRET PCR method is rapid and effective in examining a large number of snail samples simultaneously. Validation using molecular evidence from this procedure provides another tool for surveying the prevalence of O. viverrini-infected snails in Southeast Asian countries.
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Molecular evidence of Opisthorchis viverrini in infected bithyniid snails in the Lao People’s Democratic Republic by specific Hybridization Probe-based real-time fluorescence resonance energy transfer PCR method
Parasitology Research, 2011Co-Authors: Pusadee Sri-aroon, Pewpan M Intapan, Chantima Lohachit, Phunthira Phongsasakulchoti, Tongjit Thanchomnang, Viraphong Lulitanond, Alexandra Hiscox, Samlane Phompida, Pany Sananikhom, Wanchai MaleewongAbstract:Naturally occurring bithyniid snails, Bithynia siamensis goniomphalos (Prosobranchia: Bithyniidae), and their intermediate hosts were sampled from Khammouane Province, the Lao People's Democratic Republic, and the prevalence of the carcinogenic human liver fluke, Opisthorchis viverrini , was examined. The presence of O. viverrini cercariae in snails was examined by cercarial shedding test and then confirmed by specific Hybridization Probe-based real-time fluorescence resonance energy transfer (FRET) PCR method. The real-time FRET PCR method is based on a fluorescence melting curve analysis of a hybrid between an amplicon produced from the pOV-A6 specific sequence (Genbank accession no. S80278), a 162-bp repeated sequence specific to O. viverrini , and specific fluorophore-labeled Probes. Mean melting temperature of O. viverrini DNA from the cercariae and each of two positive snails by shedding test was 66.3 ± 0.1. The O. viverrini infection rate in snails was 2.47% (2/81) by cercarial shedding test but was 8.52% (4/47) by real-time FRET PCR method. The real-time FRET PCR method is rapid and effective in examining a large number of snail samples simultaneously. Validation using molecular evidence from this procedure provides another tool for surveying the prevalence of O. viverrini -infected snails in Southeast Asian countries.
Chantima Lohachit - One of the best experts on this subject based on the ideXlab platform.
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Molecular evidence of Opisthorchis viverrini in infected bithyniid snails in the Lao People's Democratic Republic by specific Hybridization Probe-based real-time fluorescence resonance energy transfer PCR method.
Parasitology Research, 2011Co-Authors: Pusadee Sri-aroon, Pewpan M Intapan, Chantima Lohachit, Phunthira Phongsasakulchoti, Tongjit Thanchomnang, Viraphong Lulitanond, Alexandra Hiscox, Samlane Phompida, Pany Sananikhom, Wanchai MaleewongAbstract:Naturally occurring bithyniid snails, Bithynia siamensis goniomphalos (Prosobranchia: Bithyniidae), and their intermediate hosts were sampled from Khammouane Province, the Lao People's Democratic Republic, and the prevalence of the carcinogenic human liver fluke, Opisthorchis viverrini, was examined. The presence of O. viverrini cercariae in snails was examined by cercarial shedding test and then confirmed by specific Hybridization Probe-based real-time fluorescence resonance energy transfer (FRET) PCR method. The real-time FRET PCR method is based on a fluorescence melting curve analysis of a hybrid between an amplicon produced from the pOV-A6 specific sequence (Genbank accession no. S80278), a 162-bp repeated sequence specific to O. viverrini, and specific fluorophore-labeled Probes. Mean melting temperature of O. viverrini DNA from the cercariae and each of two positive snails by shedding test was 66.3 ± 0.1. The O. viverrini infection rate in snails was 2.47% (2/81) by cercarial shedding test but was 8.52% (4/47) by real-time FRET PCR method. The real-time FRET PCR method is rapid and effective in examining a large number of snail samples simultaneously. Validation using molecular evidence from this procedure provides another tool for surveying the prevalence of O. viverrini-infected snails in Southeast Asian countries.
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Molecular evidence of Opisthorchis viverrini in infected bithyniid snails in the Lao People’s Democratic Republic by specific Hybridization Probe-based real-time fluorescence resonance energy transfer PCR method
Parasitology Research, 2011Co-Authors: Pusadee Sri-aroon, Pewpan M Intapan, Chantima Lohachit, Phunthira Phongsasakulchoti, Tongjit Thanchomnang, Viraphong Lulitanond, Alexandra Hiscox, Samlane Phompida, Pany Sananikhom, Wanchai MaleewongAbstract:Naturally occurring bithyniid snails, Bithynia siamensis goniomphalos (Prosobranchia: Bithyniidae), and their intermediate hosts were sampled from Khammouane Province, the Lao People's Democratic Republic, and the prevalence of the carcinogenic human liver fluke, Opisthorchis viverrini , was examined. The presence of O. viverrini cercariae in snails was examined by cercarial shedding test and then confirmed by specific Hybridization Probe-based real-time fluorescence resonance energy transfer (FRET) PCR method. The real-time FRET PCR method is based on a fluorescence melting curve analysis of a hybrid between an amplicon produced from the pOV-A6 specific sequence (Genbank accession no. S80278), a 162-bp repeated sequence specific to O. viverrini , and specific fluorophore-labeled Probes. Mean melting temperature of O. viverrini DNA from the cercariae and each of two positive snails by shedding test was 66.3 ± 0.1. The O. viverrini infection rate in snails was 2.47% (2/81) by cercarial shedding test but was 8.52% (4/47) by real-time FRET PCR method. The real-time FRET PCR method is rapid and effective in examining a large number of snail samples simultaneously. Validation using molecular evidence from this procedure provides another tool for surveying the prevalence of O. viverrini -infected snails in Southeast Asian countries.
