TY  - JOUR
AU  - Radin, Dragoslava
AU  - D'Souza, Doris H.
PY  - 2011
UR  - http://aspace.agrif.bg.ac.rs/handle/123456789/2714
AB  - Several foodborne norovirus gastroenteritis outbreaks have been linked to fresh produce. Rapid and sensitive detection can help prevent the release of contaminated produce items in the market. The objectives of this study were to apply a relatively inexpensive SYBR Green I-based real-time RT-PCR assay for the rapid detection of human norovirus (NoV) GI and GII on the surfaces of lettuce, cherry tomatoes, and green onions. Each washed produce commodity (25 g) was spiked with serial dilutions of NoV GI and GII stool samples. RNA was eluted from the produce surface and extracted using the TRIzol((TM)) method. This was followed by detection using SYBR Green I real-time RT-PCR with primers specific for NoV GI (COG1F-COG1R) and GII (COG2F-COG2R) along with an internal RNA amplification control. End-point detection limits from lettuce and tomatoes were found to be 10 RT-PCR units/25 g for GI and GII and 1 RT-PCR unit/25 g for GI and 10 RT-PCR units/25 g for GII from green onions. These results were confirmed by Tm analysis (showing peaks at 81.5 and 84A degrees C for GI and GII, respectively; and 83A degrees C for the IAC) as well as agarose gel electrophoresis that confirmed products of similar to 95 bp for GI and GII and similar to 155 bp for the RNA IAC. Results could be obtained within one working day, showing potential for routine use in diagnostics and monitoring of NoV contamination by the produce industry.
PB  - Springer, New York
T2  - Food and Environmental Virology
T1  - Simple and Rapid Detection of Human Norovirus from Produce Using SYBR Green I-based Real-time RT-PCR
EP  - 129
IS  - 3-4
SP  - 121
VL  - 3
DO  - 10.1007/s12560-011-9066-5
ER  - 

@article{
author = "Radin, Dragoslava and D'Souza, Doris H.",
year = "2011",
abstract = "Several foodborne norovirus gastroenteritis outbreaks have been linked to fresh produce. Rapid and sensitive detection can help prevent the release of contaminated produce items in the market. The objectives of this study were to apply a relatively inexpensive SYBR Green I-based real-time RT-PCR assay for the rapid detection of human norovirus (NoV) GI and GII on the surfaces of lettuce, cherry tomatoes, and green onions. Each washed produce commodity (25 g) was spiked with serial dilutions of NoV GI and GII stool samples. RNA was eluted from the produce surface and extracted using the TRIzol((TM)) method. This was followed by detection using SYBR Green I real-time RT-PCR with primers specific for NoV GI (COG1F-COG1R) and GII (COG2F-COG2R) along with an internal RNA amplification control. End-point detection limits from lettuce and tomatoes were found to be 10 RT-PCR units/25 g for GI and GII and 1 RT-PCR unit/25 g for GI and 10 RT-PCR units/25 g for GII from green onions. These results were confirmed by Tm analysis (showing peaks at 81.5 and 84A degrees C for GI and GII, respectively; and 83A degrees C for the IAC) as well as agarose gel electrophoresis that confirmed products of similar to 95 bp for GI and GII and similar to 155 bp for the RNA IAC. Results could be obtained within one working day, showing potential for routine use in diagnostics and monitoring of NoV contamination by the produce industry.",
publisher = "Springer, New York",
journal = "Food and Environmental Virology",
title = "Simple and Rapid Detection of Human Norovirus from Produce Using SYBR Green I-based Real-time RT-PCR",
pages = "129-121",
number = "3-4",
volume = "3",
doi = "10.1007/s12560-011-9066-5"
}

Radin, D.,& D'Souza, D. H.. (2011). Simple and Rapid Detection of Human Norovirus from Produce Using SYBR Green I-based Real-time RT-PCR. in Food and Environmental Virology
Springer, New York., 3(3-4), 121-129.
https://doi.org/10.1007/s12560-011-9066-5

Radin D, D'Souza DH. Simple and Rapid Detection of Human Norovirus from Produce Using SYBR Green I-based Real-time RT-PCR. in Food and Environmental Virology. 2011;3(3-4):121-129.
doi:10.1007/s12560-011-9066-5 .

Radin, Dragoslava, D'Souza, Doris H., "Simple and Rapid Detection of Human Norovirus from Produce Using SYBR Green I-based Real-time RT-PCR" in Food and Environmental Virology, 3, no. 3-4 (2011):121-129,
https://doi.org/10.1007/s12560-011-9066-5 . .

TY  - JOUR
AU  - Radin, Dragoslava
AU  - D'Souza, Doris H.
PY  - 2011
UR  - http://aspace.agrif.bg.ac.rs/handle/123456789/2632
AB  - Rapid detection of emerging virulent human noroviruses (Genogroups I and II) from clinical and food samples remains an on-going challenge. Development of internal amplification controls (IACs) in real-time RT-PCR assays to eliminate false negatives due to sample inhibition or reaction failure is critical. RNA IACs were constructed for application with two sets of previously described highly reactive degenerate primers (MON and COG) for the detection of human noroviruses (HNoVs) GI and GII. These primer sets were compared for detection sensitivity of HNoVs from outbreak stool samples (6 GI and 9 GII) by SYBR Green I based real-time reverse-transcriptase (RT)-PCR. In order to detect viruses directly from stool samples, heat release was used to expose the viral RNA (95A degrees C, 10 min). PCR conditions were optimized for each primer set before and also after IAC addition to obtain similar detection limits. Both primer sets showed equal detection limits for GII strains (4 log RT-PCR U/sample) and with one-log higher detection for GI strains (10(-5) end-point dilution; 5 log RT-PCR U/sample). The melt temperature (T (m)) of the COG and MON IAC were 83A degrees C (155 bp) and 83.5A degrees C (150 bp), respectively. Product T (m) were 84A degrees C (using MON primers for both genogroups) and 81.5A degrees C (COG for GI) and 84A degrees C (COG for GII). Agarose gel electrophoresis determined product sizes as 85 and 98 bp for GI and GII with COG, respectively, and 213 bp with MON for both genogroups. From our study, COG primers appear to have a broader detection range than the MON primers, using the 15 tested stool samples. This assay can potentially be implemented for routine HNoV detection from clinical and food samples.
PB  - Springer, New York
T2  - Food and Environmental Virology
T1  - Evaluation of Two Primer Sets Using Newly Developed Internal Amplification Controls for Rapid Human Norovirus Detection by SYBR Green I Based Real-Time RT-PCR
EP  - 69
IS  - 2
SP  - 61
VL  - 3
DO  - 10.1007/s12560-011-9057-6
ER  - 

@article{
author = "Radin, Dragoslava and D'Souza, Doris H.",
year = "2011",
abstract = "Rapid detection of emerging virulent human noroviruses (Genogroups I and II) from clinical and food samples remains an on-going challenge. Development of internal amplification controls (IACs) in real-time RT-PCR assays to eliminate false negatives due to sample inhibition or reaction failure is critical. RNA IACs were constructed for application with two sets of previously described highly reactive degenerate primers (MON and COG) for the detection of human noroviruses (HNoVs) GI and GII. These primer sets were compared for detection sensitivity of HNoVs from outbreak stool samples (6 GI and 9 GII) by SYBR Green I based real-time reverse-transcriptase (RT)-PCR. In order to detect viruses directly from stool samples, heat release was used to expose the viral RNA (95A degrees C, 10 min). PCR conditions were optimized for each primer set before and also after IAC addition to obtain similar detection limits. Both primer sets showed equal detection limits for GII strains (4 log RT-PCR U/sample) and with one-log higher detection for GI strains (10(-5) end-point dilution; 5 log RT-PCR U/sample). The melt temperature (T (m)) of the COG and MON IAC were 83A degrees C (155 bp) and 83.5A degrees C (150 bp), respectively. Product T (m) were 84A degrees C (using MON primers for both genogroups) and 81.5A degrees C (COG for GI) and 84A degrees C (COG for GII). Agarose gel electrophoresis determined product sizes as 85 and 98 bp for GI and GII with COG, respectively, and 213 bp with MON for both genogroups. From our study, COG primers appear to have a broader detection range than the MON primers, using the 15 tested stool samples. This assay can potentially be implemented for routine HNoV detection from clinical and food samples.",
publisher = "Springer, New York",
journal = "Food and Environmental Virology",
title = "Evaluation of Two Primer Sets Using Newly Developed Internal Amplification Controls for Rapid Human Norovirus Detection by SYBR Green I Based Real-Time RT-PCR",
pages = "69-61",
number = "2",
volume = "3",
doi = "10.1007/s12560-011-9057-6"
}

Radin, D.,& D'Souza, D. H.. (2011). Evaluation of Two Primer Sets Using Newly Developed Internal Amplification Controls for Rapid Human Norovirus Detection by SYBR Green I Based Real-Time RT-PCR. in Food and Environmental Virology
Springer, New York., 3(2), 61-69.
https://doi.org/10.1007/s12560-011-9057-6

Radin D, D'Souza DH. Evaluation of Two Primer Sets Using Newly Developed Internal Amplification Controls for Rapid Human Norovirus Detection by SYBR Green I Based Real-Time RT-PCR. in Food and Environmental Virology. 2011;3(2):61-69.
doi:10.1007/s12560-011-9057-6 .

Radin, Dragoslava, D'Souza, Doris H., "Evaluation of Two Primer Sets Using Newly Developed Internal Amplification Controls for Rapid Human Norovirus Detection by SYBR Green I Based Real-Time RT-PCR" in Food and Environmental Virology, 3, no. 2 (2011):61-69,
https://doi.org/10.1007/s12560-011-9057-6 . .