A one-step multiplex reverse transcription PCR (RT-PCR) method comprising six primer sets (for the detection of norovirus GI and GII, hepatitis A virus, rotavirus, and astrovirus) was developed to simultaneously detect four kinds of pathogenic viruses. The size of the PCR products for norovirus GI a...
A one-step multiplex reverse transcription PCR (RT-PCR) method comprising six primer sets (for the detection of norovirus GI and GII, hepatitis A virus, rotavirus, and astrovirus) was developed to simultaneously detect four kinds of pathogenic viruses. The size of the PCR products for norovirus GI and GII, hepatitis A virus (VP3/VP1 and P2A regions), rotavirus, and astrovirus were 330, 164, 244, 198, 629, and 449 bp, respectively. The RT-PCR with the six primer sets showed specificity for the pathogenic viruses. The detection limit of the developed multiplex RT-PCR, as evaluated using serially diluted viral RNAs, was comparable to that of one-step single RT-PCR. Moreover, this multiplex RT-PCR was evaluated using food samples such as water, oysters, lettuce, and vegetable product. These food samples were artificially spiked with the four kinds of viruses in diverse combinations, and the spiked viruses in all food samples were detected successfully.
A one-step multiplex reverse transcription PCR (RT-PCR) method comprising six primer sets (for the detection of norovirus GI and GII, hepatitis A virus, rotavirus, and astrovirus) was developed to simultaneously detect four kinds of pathogenic viruses. The size of the PCR products for norovirus GI and GII, hepatitis A virus (VP3/VP1 and P2A regions), rotavirus, and astrovirus were 330, 164, 244, 198, 629, and 449 bp, respectively. The RT-PCR with the six primer sets showed specificity for the pathogenic viruses. The detection limit of the developed multiplex RT-PCR, as evaluated using serially diluted viral RNAs, was comparable to that of one-step single RT-PCR. Moreover, this multiplex RT-PCR was evaluated using food samples such as water, oysters, lettuce, and vegetable product. These food samples were artificially spiked with the four kinds of viruses in diverse combinations, and the spiked viruses in all food samples were detected successfully.
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제안 방법
After artificially inoculating viruses into samples that tested negative for each virus, the samples were handled with different methods depending on the sample type. All of the procedures were conducted according to the recommended guidelines established by the MFDS for the detection of foodborne viruses, with minor modifications.
In Korea, a less fermented vegetable product is infrequently associated with acute gastroenteritis in humans, likely due to the use of untreated groundwater [9]. Focusing on these foods, we evaluated whether our multiplex PCR method could be applied. All of the samples tested were analyzed successfully.
For this reason, conventional RT-PCR methods are still required for a thorough sequence analysis. In this study, to make it possible to do direct sequencing on the multiplex RT-PCR amplicons, a one-step multiplex RT-PCR method was developed with NoV GI, NoV GII, HAV, AstV, and RoV. On the basis of existing primer sets, specific primer pairs were selected, and some primer sets were redesigned to prevent PCR amplicons from overlapping (and to increase sensitivity).
One-step single RT-PCR was performed with 5 µl of total extracted RNA, 4 µl of dNTPs (each dNTP at 2.5 mM; Bioneer, Korea), 5 µl of 10× reaction buffer, 1 µl of Top DNA polymerase (5 U/µl; Bioneer), 1 µl of avian myeloblastosis virus (AMV) reverse transcriptase (10 U/µl; Progema, USA), 1 µl of each forward/reverse primer, and DEPC-treated water in a final reaction volume of 50 µl.
The developed assay was applied to water, oysters, lettuce, and vegetable product samples that were artificially inoculated with the viruses. After the inoculated samples were processed (using different methods, depending on the sample type), the resulting solutions were used to extract viral RNA.
After the inoculated samples were processed (using different methods, depending on the sample type), the resulting solutions were used to extract viral RNA. The extracts were then tested using the developed method. All samples yielded specific detection with the expected amplicon for the relevant virus (Fig.
Primers named MON269 and MON270 were used for amplifying AstV, generating an amplicon size of 449 bp [19]. The two primer sets for NoV GII and HAV (P2A region) were newly designed in this study using the Primer Designer program, ver. 3.0 (Scientific and Educational Software, USA) based on conserved sequences of reference strains of NoV and HAV obtained from the GenBank database. These two pairs of primers generated 164 bp and 198 bp PCR amplicons for NoV GII and HAV, respectively.
To compare the detection limits of the one-step single and multiplex RT-PCR methods, 10-fold serial dilutions (100 to 10-5) of five different viral RNA samples in DEPC-treated water were tested using both assays. In the one-step single RT-PCR, the PCR mixture conditions were the same as those of the one-step multiplex RT-PCR except that a single, specific primer set for each target virus was contained in a separate reaction tube.
대상 데이터
In this study, four kinds of RNA viruses (NoV GI and GII, HAV, AstV, and RoV) were selected because of their frequent contributions to viral food poisoning in Korea. A total of six primer sets for these four viruses were selected, designed to amplify specific target regions.
NoV GI, NoV GII, and AstV were provided by the Waterborne Virus Bank at Catholic University in Korea. The HAV HM175 strain, grown in fetal rhesus kidney-derived (FRhK-4) cells, and group A human RoV, grown in a monkey kidney cell line (MA104), were obtained from the Ministry of Food and Drug Safety (MFDS) in Korea.
Six sets of primers for specific viruses were used in this study (Table 1). All of the primer sets targeted a gene for the capsid protein of the relevant virus.
후속연구
Based on these results, this assay has the potential to serve as an effective diagnostic tool for identifying four kinds of RNA viruses (norovirus, hepatitis A virus, rotavirus, and astrovirus) that cause gastroenteritis. The multiplex RT-PCR assay developed in this study could be a reliable method for the rapid, accurate, and simultaneous analysis of foodborne viruses relevant to food safety.
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