Detection of viable murine norovirus using the plaque assay and propidium-monoazide-combined real-time reverse transcription-polymerase chain reaction.
Human norovirus (HuNoV) is the most common cause of gastroenteritis worldwide. The lack of a virus culture system makes it difficult to determine the viability of norovirus by only reverse transcription-polymerase chain reaction (RT-PCR) or real-time quantitative RT-PCR (qRT-PCR). The aim of this study was to investigate the detection of viable murine norovirus (MNV) by combining propidium monoazide (PMA) or ethidium monoazide (EMA) with qRT-PCR. MNV (5.21log10PFU/mL) was subjected to heat treatment at room temperature, 65, 70, 75, 80, 85, or 90°C in a water bath for 1min. The plaque assay, qRT-PCR, PMA-combined qRT-PCR, and EMA-combined qRT-PCR were then performed with heat exposed MNV samples. The MNV titer was reduced by 0.38, 1.34, and 3.71log10PFU/mL at temperatures of 65, 70, and 75°C, respectively. MNV was reduced >4.21log10PFU/mL at 80, 85, and 90°C heat inactivation. PMA (EMA) value equation for the interpretation of the viability of MNV was derived as follows: PMA (EMA) value=-logRN-logRP (RN: the relative quantity value of the not-treated sample, and RP: the relative quantity value of the PMA- or EMA-treated sample as determined by qRT-PCR). By PMA-combined qRT-PCR, the viable PMA value was 0.32, 0.83, and 2.62 for the 65, 70, and 75°C preheated MNVs, respectively. The viable PMA values for the viruses heated at 80, 85, and 90°C were all greater than 3.0, which was the cutoff value for discriminating between live and dead MNVs. The results of EMA-combined qRT-PCR were similar to those of qRT-PCR. Thus, PMA-combined qRT-PCR correlated well with the plaque assay in detecting viable MNVs.
Department of Food and Nutrition, Chung-Ang University, Anseong, Gyeonggi 456-756, South Korea.
Minhwa
M
Lee
Department of Food and Nutrition, Chung-Ang University, Anseong, Gyeonggi 456-756, South Korea.
Dong Joo
DJ
Seo
Department of Food and Nutrition, Chung-Ang University, Anseong, Gyeonggi 456-756, South Korea.
Jina
J
Seo
Department of Food and Nutrition, Chung-Ang University, Anseong, Gyeonggi 456-756, South Korea.
Hyejin
H
Oh
Department of Food and Nutrition, Chung-Ang University, Anseong, Gyeonggi 456-756, South Korea.
Su Been
SB
Jeon
School of Food Science and Technology, Chung-Ang University, Anseong, Gyeonggi 456-756, South Korea.
Sang-Do
SD
Ha
Korea Zoonosis Research Institute, Chonbuk National University, Deokjin-dong 1ga, Deokjin-gu, Jeonju-si, Jeollabuk-do, South Korea.
Jinjong
J
Myoung
Department of Infectious Disease, College of Veterinary Medicine, Konkuk University, Seoul, South Korea.
In-Soo
IS
Choi
Department of Food and Nutrition, Chung-Ang University, Anseong, Gyeonggi 456-756, South Korea; School of Food Science and Technology, Chung-Ang University, Anseong, Gyeonggi 456-756, South Korea. Electronic address: cchoi@cau.ac.kr.
Changsun
C
Choi
D000818:Animals; D001386:Azides / Q000378:metabolism; D002460:Cell Line; D008264:Macrophages / Q000821:virology; D051379:Mice; D050296:Microbial Viability; D029322:Norovirus / Q000302:isolation & purification* / Q000502:physiology*; D011419:Propidium / Q000031:analogs & derivatives / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature; D010948:Viral Plaque Assay / Q000379:methods*; D014773:Virology / Q000379:methods*
D001386:Azides; C533957:propidium monoazide; D011419:Propidium; C014535:8-azidoethidium
Ethidium monoazide; Murine norovirus; Plaque assay; Propidium monoazide; Viable