Assay application food pcr inva

Application of in situ loop-mediated isothermal DeepDyve

Detection of Some SalmonellaEnteritidisVirulence Genes by

inva pcr assay food application

Detection of invasion gene invA in Salmonella spp. 6-11-2019 · SureTect Pathogen Detection Kits. Filter Products [5] H7 PCR Assay. SureTect™ Listeria monocytogenes PCR Assay (Thermo Scientific™) Detect Listeria monocytogenes in even the most challenging food types with this multiplex real-time assay that provides highly-specific detection of the prfA gene., PDF The objective of this study was to develop and evaluate a SYBR Green 1 real-time PCR method for the specific detection of Salmonella spp. in dairy farm environmental samples. Previously reported 119-bp invA gene was selected for ….

Loop-Mediated Isothermal Amplification for Salmonella

InvA gene specific PCR for detection of Salmonella from. Aims: Development of a PCR assay that can target multiple genes for rapid detection of Salmonella enterica serovar Typhi (S. Typhi) from water and food samples. Methods and Results: PCR primers for invasion, O, H and Vi antigen genes, invA, prt, fliC‐d and viaB were designed and used for the rapid detection of S. Typhi by multiplex PCR., ovel Simplex Real-Time PCR Assay for Rapid Molecular A N Detection and Typing of Salmonella Typhimurium We first tested a series of Salmonella serotypes, including Typhimurium as well as some of the most relevant serotypes for the food industry according to data recorded from recent outbreaks in the US (Jackson et al., 2013)..

In order to assess the detection sensitivity of the multiplex PCR assay for its application to food samples, three type of food samples including vegetable, chicken and raw meat pork were inoculated with different concentrations of three foodborne pathogens and incubated for 12, 18, and 24 h in SEB enrichment medium. Application of SYBR green real-time PCR assay for specific detection of Salmonella spp. in dairy farm environmental samples Hyang-Mi Nam, Velusamy Srinivasan, Barbara E. Gillespie, Shelton E. Murinda, Stephen P. OliverT Food Safety Centerof Excellence and To validate the real-time PCR assay, an experiment was conducted with both spiked and

Department of Food Hygiene, Faculty of Veterinary Medicine, Aswan University, 81528 Aswan, Egypt Abstract: Detection of pathogenic Salmonella isolated from meat and poultry products by detecting virulence invA gene using PCR technique.A total of100 meat and poultry products samples were collected from shops ABSTRACT A multiplex PCR was developed to identify the two most common serovars of Salmonella causing foodborne illness in Canada, Home > Journal of Food Protection > February 2017 > Evaluation of a Multiplex PCR Assay for the Identification of …

The bound bacteria were eluted and tested with PCR targeting the invA gene of Salmonella. Ten different serovars of Salmonella enterica and four non-Salmonella were tested to determine the specificity of the DNA aptamer. For field application, 14 different food samples were tested and compared with the culture method. The objective of this study was to develop and evaluate a SYBR Green 1 real-time PCR method for the specific detection of Salmonella spp. in dairy farm environmental samples. Previously reported 119-bp invA gene was selected for specificity, and 124 Salmonella spp. including type strains and 116 non-Salmonella strains were evaluated.

Application of SYBR green real-time PCR assay for specific detection of Salmonella spp. in dairy farm environmental samples. International Journal of Food Microbiology, 2005. Velusamy Srinivasan. S. Oliver. Shelton Murinda. Velusamy Srinivasan. S. Oliver. Shelton Murinda. Inv A gene specific PCR for detection of Salmonella from broilers at 370C for 24 hrs. The biochemical characters of project, showed high selectivity on 242 Salmonella bacteria from non-lactose fermenting determined strains (sensitivity 99.6%) and 122 non-Salmonella using triple sugar iron agar (Himedia).

PDF The objective of this study was to develop and evaluate a SYBR Green 1 real-time PCR method for the specific detection of Salmonella spp. in dairy farm environmental samples. Previously reported 119-bp invA gene was selected for … 1-6-2009 · Only the bagged spinach preenrichment, spiked with live cells, gave a positive signal by the qRT-PCR assay reported here. This clearly showed that the qRT-PCR assay, based on invA mRNA detection, has great potential to be used as a viability marker for Salmonella cells in …

Application of SYBR green real-time PCR assay for specific detection of Salmonella spp. in dairy farm environmental samples Hyang-Mi Nam, Velusamy Srinivasan, Barbara E. Gillespie, Shelton E. Murinda, Stephen P. OliverT Food Safety Centerof Excellence and To validate the real-time PCR assay, an experiment was conducted with both spiked and 24-9-2016В В· Application of loop-mediated isothermal amplification with propidium monoazide treatment to detect live Salmonella in chicken carcasses Design of LAMP and PCR Assay Primers. The Salmonella invasion gene (invA; CFU/reaction). In contrast, the invA-based PCR had a detection limit of 1.0 Г— 10 4 CFU/mL

Aims: Development of a PCR assay that can target multiple genes for rapid detection of Salmonella enterica serovar Typhi (S. Typhi) from water and food samples. Methods and Results: PCR primers for invasion, O, H and Vi antigen genes, invA, prt, fliC‐d and viaB were designed and used for the rapid detection of S. Typhi by multiplex PCR. Application of SYBR green real-time PCR assay for specific detection of Salmonella spp. in dairy farm environmental samples Hyang-Mi Nam, Velusamy Srinivasan, Barbara E. Gillespie, Shelton E. Murinda, Stephen P. OliverT Food Safety Centerof Excellence and To validate the real-time PCR assay, an experiment was conducted with both spiked and

The PCR technique confirmed that all Salmonella isolates carried the invA gene (DNA amplification showed one distinct band with molecular weight of 506 bp amplified fragment on electrophoresis in agarose gel).The PCR assay described herein was found to be a rapid and simple method to confirm the isolates as Salmonella. Read "Application of in situ loop-mediated isothermal amplification method for detection of Salmonella in foods, Food Control" on DeepDyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips.

Establishment and preliminary application of LAMP invA gene assay for rapid detection of Salmonella 5’-Nuclease PCR Assays for Foodborne Pathogen Detection using the Agilent-Stratagene Mx3000P Q-PCR System Application: Food Safety Introduction Foodborne disease is estimated to cause 76 million illnesses and 5,000 deaths annually in the United States [1]. Of the known pathogens that cause foodborne

application of Salmonella LAMP assays in a wide array of food and feed matrices. Recent progress in assay design, platform development, commercial application, and method validation is reviewed. Future perspectives toward more practical and wider applications of Salmonella LAMP assays in food and feed testing are discussed. realв€’time PCR assay was developed to detect Salmonella in sprout irrigation water. Specific detection was achieved by targeting a r egion of the invasion gene, invA. The detection limit for Salmonella in sterile water was approximately 400 CFU, and in sprout irrigation water the detection limit was approximately 200 CFU.

SureTect Pathogen Detection Kits Products and Microbial. internationally validated conventional PCR system targeting the invA gene. This PCR assay was suggested as a standard method for the detection of Salmonella spp. in the FOOD-PCR project [2]. The aim of this study was to follow the contamination of food of animal origin with Salmonella spp. by Step One real-time PCR. 2. Materials and methods, To detect Salmonella in foods, a SYBR Green в…  real-time PCR assay was established. Primers specific for the invA gene in Salmonella were selected to conduct the SYBR Green в…  real-time PCR assays. The test for specificity was performed with Salmonella and non-homologous strains. The reproducibility test was based on different species of.

Inv A gene specific PCR for detection of Salmonella from

inva pcr assay food application

Potential Use of DNA Aptamer-Magnetic Bead Separation-PCR. application of Salmonella LAMP assays in a wide array of food and feed matrices. Recent progress in assay design, platform development, commercial application, and method validation is reviewed. Future perspectives toward more practical and wider applications of Salmonella LAMP assays in food and feed testing are discussed., 23-3-2004 · A robust 5′ nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5..

(PDF) Selective Amplification of prt tyv and invA Genes. For field application, 14 different food samples were tested and com-pared with the culture method. Results: The limit of detection of AMS-PCR method was 102 CFU/ml which was 10 times more sensitive than conventional PCR without AMS (103 CFU/ml). The AMS-PCR assay showed high specificity as it detected ten different serovars of Salmonella enterica, salmonellae isolates. Application of the PCR assay on Salmonella in shrimp after the selective enrichment step suggested that all 16 samples were positive for Salmonella. At the same time, the conventional method could only detected 3 samples for Salmonella positive. Keywords: invA gene, optimization, PCR , Salmonella spp., shrimp ABSTRAK.

Application of loop-mediated isothermal amplification with

inva pcr assay food application

(PDF) Application of SYBR green real-time PCR assay for. Inv A gene specific PCR for detection of Salmonella from broilers at 370C for 24 hrs. The biochemical characters of project, showed high selectivity on 242 Salmonella bacteria from non-lactose fermenting determined strains (sensitivity 99.6%) and 122 non-Salmonella using triple sugar iron agar (Himedia). PDF The objective of this study was to develop and evaluate a SYBR Green 1 real-time PCR method for the specific detection of Salmonella spp. in dairy farm environmental samples. Previously reported 119-bp invA gene was selected for ….

inva pcr assay food application

  • Evaluation of a Multiplex PCR Assay for the Identification
  • Detection of Some SalmonellaEnteritidisVirulence Genes by
  • Establishment and preliminary application of LAMP invA

  • 3-2-2011В В· Methods and Results: A novel rapid and simple isothermal target and probe amplification (iTPA) assay that rapidly amplifies target DNA (Salmonella invA gene) using a FRET‐based signal probe in an isothermal environment was developed for detection Salmonella spp. in pre‐enriched food samples. The assay was able to specifically detect all of Detection of the enterotoxins A, B and C genes in Therefore, an interesting development of m-PCR assay staphylococcus aureus from goat and bovine in this study would be its application directly on milk and mastitis in Brazilian dairy herds. Vet. Microbiol., 106: …

    Aims: Development of a PCR assay that can target multiple genes for rapid detection of Salmonella enterica serovar Typhi (S. Typhi) from water and food samples. Methods and Results: PCR primers for invasion, O, H and Vi antigen genes, invA, prt, fliC‐d and viaB were designed and used for the rapid detection of S. Typhi by multiplex PCR. Detection of the enterotoxins A, B and C genes in Therefore, an interesting development of m-PCR assay staphylococcus aureus from goat and bovine in this study would be its application directly on milk and mastitis in Brazilian dairy herds. Vet. Microbiol., 106: …

    Application of SYBR green real-time PCR assay for specific detection of Salmonella spp. in dairy farm environmental samples. Author links open overlay panel Hyang-Mi Nam Velusamy Srinivasan Barbara E. Gillespie Shelton E. Murinda Stephen P. Oliver. Show more. The bound bacteria were eluted and tested with PCR targeting the invA gene of Salmonella. Ten different serovars of Salmonella enterica and four non-Salmonella were tested to determine the specificity of the DNA aptamer. For field application, 14 different food samples were tested and compared with the culture method.

    Development and Evaluation of a Multiplex PCR for Simultaneous Detection of Five Foodborne Pathogens. Thuy Trang Nguyen 1,3#, we developed and evaluated a multiplex PCR for simultaneous detection of the five food borne pathogens In order to assess the detection sensitivity of the multiplex PCR assay for its application to food The bound bacteria were eluted and tested with PCR targeting the invA gene of Salmonella. Ten different serovars of Salmonella enterica and four non-Salmonella were tested to determine the specificity of the DNA aptamer. For field application, 14 different food samples were tested and compared with the culture method.

    The objective of this study was to develop and evaluate a SYBR Green 1 real-time PCR method for the specific detection of Salmonella spp. in dairy farm environmental samples. Previously reported 119-bp invA gene was selected for specificity, and 124 Salmonella spp. including type strains and 116 non-Salmonella strains were evaluated. To detect Salmonella in foods, a SYBR Green в…  real-time PCR assay was established. Primers specific for the invA gene in Salmonella were selected to conduct the SYBR Green в…  real-time PCR assays. The test for specificity was performed with Salmonella and non-homologous strains. The reproducibility test was based on different species of

    salmonellae isolates. Application of the PCR assay on Salmonella in shrimp after the selective enrichment step suggested that all 16 samples were positive for Salmonella. At the same time, the conventional method could only detected 3 samples for Salmonella positive. Keywords: invA gene, optimization, PCR , Salmonella spp., shrimp ABSTRAK Read "Application of in situ loop-mediated isothermal amplification method for detection of Salmonella in foods, Food Control" on DeepDyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips.

    23-3-2004 · A robust 5′ nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5. 1-6-2009 · Only the bagged spinach preenrichment, spiked with live cells, gave a positive signal by the qRT-PCR assay reported here. This clearly showed that the qRT-PCR assay, based on invA mRNA detection, has great potential to be used as a viability marker for Salmonella cells in …

    application of Salmonella LAMP assays in a wide array of food and feed matrices. Recent progress in assay design, platform development, commercial application, and method validation is reviewed. Future perspectives toward more practical and wider applications of Salmonella LAMP assays in food and feed testing are discussed. 23-3-2004 · A robust 5′ nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5.

    inva pcr assay food application

    PCR, on the other hand, is generally susceptible to various assay inhibitors present in complex food or feed matrices (Abu Al-Soud and Radstrom, 2000; Maciorowski et al., 2005). LAMP is also more versatile in terms of amplicon detection methods, which include naked eye, colorimetry, turbidity, fluorescence, and bioluminescence, among many others (Zhang et al. , 2014 ). The bound bacteria were eluted and tested with PCR targeting the invA gene of Salmonella. Ten different serovars of Salmonella enterica and four non-Salmonella were tested to determine the specificity of the DNA aptamer. For field application, 14 different food samples were tested and compared with the culture method.

    Development of a Real-Time PCR Assay to Specifically. journal of food and nutrition research, 2(6), 294-300. ateba, collins njie, and biotumelo mochaiwa. "use of inva gene specific pcr analysis for the detection of virulent salmonella species in beef products in the north west province, south africa." journal of food and nutrition research 2, no. 6 (2014): 294-300., ries to reduce the time required for identiffication of food- or waterborne pathogens. real-time pcr is currently used for the diagnosis of infectious agents, and there are few reports of the application of real-time pcr for the direct detection of escherichia coli strain o157:h7 (11) and plesiomonas shig-elloides (20) in stool specimens.).

    Detection of the enterotoxins A, B and C genes in Therefore, an interesting development of m-PCR assay staphylococcus aureus from goat and bovine in this study would be its application directly on milk and mastitis in Brazilian dairy herds. Vet. Microbiol., 106: … ries to reduce the time required for identification of food- or waterborne pathogens. Real-time PCR is currently used for the diagnosis of infectious agents, and there are few reports of the application of real-time PCR for the direct detection of Escherichia coli strain O157:H7 (11) and Plesiomonas shig-elloides (20) in stool specimens.

    Application of SYBR green real-time PCR assay for specific detection of Salmonella spp. in dairy farm environmental samples Hyang-Mi Nam, Velusamy Srinivasan, Barbara E. Gillespie, Shelton E. Murinda, Stephen P. OliverT Food Safety Centerof Excellence and To validate the real-time PCR assay, an experiment was conducted with both spiked and application of Salmonella LAMP assays in a wide array of food and feed matrices. Recent progress in assay design, platform development, commercial application, and method validation is reviewed. Future perspectives toward more practical and wider applications of Salmonella LAMP assays in food and feed testing are discussed.

    1-6-2018В В· This review provides an overview of international efforts in the past decade on the development and application of Salmonella LAMP assays in a wide array of food and feed matrices. Recent progress in assay design, platform development, commercial application, and method validation is reviewed. For field application, 14 different food samples were tested and com-pared with the culture method. Results: The limit of detection of AMS-PCR method was 102 CFU/ml which was 10 times more sensitive than conventional PCR without AMS (103 CFU/ml). The AMS-PCR assay showed high specificity as it detected ten different serovars of Salmonella enterica

    InvA gene specific PCR for detection of Salmonella from boilers. Inv A gene specific PCR for detection of Salmonella from broilers. S. Weybridge and S. Naestved have one isolate (6.25%) .The application of PCR for detection of invA, avrA and stn virulence genes in nine salmonella serovars revealed that these genes (invA, 6-11-2019В В· SureTect Pathogen Detection Kits. Filter Products [5] H7 PCR Assay. SureTectв„ў Listeria monocytogenes PCR Assay (Thermo Scientificв„ў) Detect Listeria monocytogenes in even the most challenging food types with this multiplex real-time assay that provides highly-specific detection of the prfA gene.

    Journal of Food and Nutrition Research, 2(6), 294-300. Ateba, Collins Njie, and Biotumelo Mochaiwa. "Use of invA Gene Specific PCR Analysis for the Detection of Virulent Salmonella Species in Beef Products in the North West Province, South Africa." Journal of Food and Nutrition Research 2, no. 6 (2014): 294-300. 1-11-2003В В· A duplex real-time SYBR Green LightCycler PCR (LC-PCR) assay with DNA extraction using the QIAamp DNA Stool Mini kit was evaluated with regard to detection of 8 of 17 species of food- or waterborne pathogens in five stool specimens in 2 h or less. The protocol used the same LC-PCR with 20 pairs of specific primers. The products formed were

    A combined enrichment/ newly developed invA TaqMan® real-time PCR (qPCR) method as a screening assay to detect Salmonella spp. in 500 naturally food matrices is evaluated. DNA template for qPCR was extracted from an overnight pre-enriched sample in buffered peptone water using lysis–guanidine isothiocyanate method. Heterologous internal salmonellae isolates. Application of the PCR assay on Salmonella in shrimp after the selective enrichment step suggested that all 16 samples were positive for Salmonella. At the same time, the conventional method could only detected 3 samples for Salmonella positive. Keywords: invA gene, optimization, PCR , Salmonella spp., shrimp ABSTRAK

    inva pcr assay food application

    Establishment and preliminary application of LAMP invA

    Potential Use of DNA Aptamer-Magnetic Bead Separation-PCR. detection of the enterotoxins a, b and c genes in therefore, an interesting development of m-pcr assay staphylococcus aureus from goat and bovine in this study would be its application directly on milk and mastitis in brazilian dairy herds. vet. microbiol., 106: вђ¦, detection of the enterotoxins a, b and c genes in therefore, an interesting development of m-pcr assay staphylococcus aureus from goat and bovine in this study would be its application directly on milk and mastitis in brazilian dairy herds. vet. microbiol., 106: вђ¦).

    inva pcr assay food application

    Screening and Detecting Salmonella in Different Food

    Application of in situ loop-mediated isothermal DeepDyve. 24-9-2016в в· application of loop-mediated isothermal amplification with propidium monoazide treatment to detect live salmonella in chicken carcasses design of lamp and pcr assay primers. the salmonella invasion gene (inva; cfu/reaction). in contrast, the inva-based pcr had a detection limit of 1.0 г— 10 4 cfu/ml, ovel simplex real-time pcr assay for rapid molecular a n detection and typing of salmonella typhimurium we first tested a series of salmonella serotypes, including typhimurium as well as some of the most relevant serotypes for the food industry according to data recorded from recent outbreaks in the us (jackson et al., 2013).).

    inva pcr assay food application

    Multiplex PCR for simultaneous identification of E. coli

    Detection of invasion gene invA in Salmonella spp. abstract a multiplex pcr was developed to identify the two most common serovars of salmonella causing foodborne illness in canada, home > journal of food protection > february 2017 > evaluation of a multiplex pcr assay for the identification of вђ¦, to avoid bias, all the 96 isolates were screened for the salmonella specific inva gene through pcr analysis and only 10 (10.4%) isolates were positively identified. moreover, none of the isolates possessed the flic flagella gene while a small proportion 11(11.5%) were positive for the fljb gene fragments.).

    inva pcr assay food application

    Detection of Live Salmonella sp. Cells in Produce by a

    Rapid and simple detection of the invA gene in Salmonella. detection of the enterotoxins a, b and c genes in therefore, an interesting development of m-pcr assay staphylococcus aureus from goat and bovine in this study would be its application directly on milk and mastitis in brazilian dairy herds. vet. microbiol., 106: вђ¦, to avoid bias, all the 96 isolates were screened for the salmonella specific inva gene through pcr analysis and only 10 (10.4%) isolates were positively identified. moreover, none of the isolates possessed the flic flagella gene while a small proportion 11(11.5%) were positive for the fljb gene fragments.).

    inva pcr assay food application

    Recent advances in quantitative PCR (qPCR) applications in

    Detection of Live Salmonella sp. Cells in Produce by a. ries to reduce the time required for identiffication of food- or waterborne pathogens. real-time pcr is currently used for the diagnosis of infectious agents, and there are few reports of the application of real-time pcr for the direct detection of escherichia coli strain o157:h7 (11) and plesiomonas shig-elloides (20) in stool specimens., development and evaluation of a multiplex pcr for simultaneous detection of five foodborne pathogens. thuy trang nguyen 1,3#, we developed and evaluated a multiplex pcr for simultaneous detection of the five food borne pathogens in order to assess the detection sensitivity of the multiplex pcr assay for its application to food).

    3-2-2011 · Methods and Results: A novel rapid and simple isothermal target and probe amplification (iTPA) assay that rapidly amplifies target DNA (Salmonella invA gene) using a FRET‐based signal probe in an isothermal environment was developed for detection Salmonella spp. in pre‐enriched food samples. The assay was able to specifically detect all of internationally validated conventional PCR system targeting the invA gene. This PCR assay was suggested as a standard method for the detection of Salmonella spp. in the FOOD-PCR project [2]. The aim of this study was to follow the contamination of food of animal origin with Salmonella spp. by Step One real-time PCR. 2. Materials and methods

    Detection of the enterotoxins A, B and C genes in Therefore, an interesting development of m-PCR assay staphylococcus aureus from goat and bovine in this study would be its application directly on milk and mastitis in Brazilian dairy herds. Vet. Microbiol., 106: … PDF The objective of this study was to develop and evaluate a SYBR Green 1 real-time PCR method for the specific detection of Salmonella spp. in dairy farm environmental samples. Previously reported 119-bp invA gene was selected for …

    Establishment and preliminary application of LAMP invA gene assay for rapid detection of Salmonella The objective of this study was to develop and evaluate a SYBR Green 1 real-time PCR method for the specific detection of Salmonella spp. in dairy farm environmental samples. Previously reported 119-bp invA gene was selected for specificity, and 124 Salmonella spp. including type strains and 116 non-Salmonella strains were evaluated.

    Department of Food Hygiene, Faculty of Veterinary Medicine, Aswan University, 81528 Aswan, Egypt Abstract: Detection of pathogenic Salmonella isolated from meat and poultry products by detecting virulence invA gene using PCR technique.A total of100 meat and poultry products samples were collected from shops ries to reduce the time required for identification of food- or waterborne pathogens. Real-time PCR is currently used for the diagnosis of infectious agents, and there are few reports of the application of real-time PCR for the direct detection of Escherichia coli strain O157:H7 (11) and Plesiomonas shig-elloides (20) in stool specimens.

    1-6-2018В В· This review provides an overview of international efforts in the past decade on the development and application of Salmonella LAMP assays in a wide array of food and feed matrices. Recent progress in assay design, platform development, commercial application, and method validation is reviewed. To detect Salmonella in foods, a SYBR Green в…  real-time PCR assay was established. Primers specific for the invA gene in Salmonella were selected to conduct the SYBR Green в…  real-time PCR assays. The test for specificity was performed with Salmonella and non-homologous strains. The reproducibility test was based on different species of

    Application of magnetic immuno-polymerase chain reaction assay for detection of Salmonella spp. in chicken meats. The IMS and PCR assay combines selective extraction of Salmonella by specific antibodies with primer-specific (primer pair based on the sequence of invA … another PCR assay can detect Salmonella enterica subspecies enterica serovar Enteritidis (e.g. Applied Biosystems ™ MicroSEQ Salmonella spp. Detection Kit versus TaqMan™ Salmonella Enteritidis Detection Kit). Improve your poultry testing by going molecular APPLICATION NOTE: Pathogen testing and typing kits food safety

    inva pcr assay food application

    (PDF) Application of SYBR green real-time PCR assay for