Arch Virol (2017) 162:3389–3396 DOI 10.1007/s00705-017-3505-z

ORIGINAL ARTICLE

Detection of waterborne norovirus genogroup I strains using an improved real time RT‑PCR assay Han‑Gil Cho1 · Sung‑Geun Lee2 · Su‑Kyoung Mun1 · Myung‑Jin Lee1 · Po‑Hyun Park1 · Weon‑Hwa Jheong3 · Mi‑Hye Yoon1 · Soon‑Yong Paik4 

Received: 19 February 2017 / Accepted: 23 June 2017 / Published online: 4 August 2017 © Springer-Verlag GmbH Austria 2017

Abstract  Noroviruses (NoVs) are the major global source of acute gastroenteritis (AGE) outbreaks. To detect NoVs, real-time reverse transcription-quantitative PCR (RT-qPCR) assays have been widely employed since the first decade of the 2­ 1st century. We developed a redesigned probe, JJV1PM, for RT-qPCR assay detection of NoV genogroup (G) I strains. The new RT-qPCR assay using the JJV1PMprobe showed broader strain reactivity for 10 NoV GI genotypes, while the old method, using the JJV1PT-probe assay, detected only 7 NoV GI genotypes in a validation panel using human fecal specimens. The improved RTqPCR assay was also successfully applied to water samples. The JJV1PM-probe assay identified 7 NoV GI genotypes, whereas the JJV1PT-probe assay detected only 2 NoV GI genotypes from water samples. Notably, groundwater-borne NoV GI strains detected by the improved JJV1PM-probe assay were associated with groundwater-borne AGE outbreaks in South Korea. The results of this study underscore the importance of the evaluation of RT-qPCR assays using recently circulating NoV strains prior to field application.

* Soon‑Yong Paik [email protected] 1



Division of Public Health Research, Gyeonggi Province Institute of Health and Environment, Suwon, Republic of Korea

2



Korea Zoonosis Research Institute, Chonbuk National University, Iksan, Republic of Korea

3



Environmental Infrastructure Research Department, National Institute of Environmental Research, Incheon, Republic of Korea

4



Department of Microbiology, College of Medicine, The Catholic University of Korea, 222, Banpo‑daero, Seocho‑gu, Seoul 06591, Republic of Korea

Abbreviations NoV Norovirus AGE Acute Gastroenteritis RT-qPCR Real-Time Reverse Transcription-Quantitative PCR ORF Open Reading Frames

Introduction Noroviruses (NoVs) are the major etiological agents of acute viral gastroenteritis (AGE) outbreaks worldwide [1]. In the USA, NoV causes 570–800 deaths, 56,000–71,000 hospitalizations, 400,000 emergency room visits, and 1.7–1.9 million outpatient visits annually [2]. NoVs are genetically diverse single-stranded RNA viruses belonging to the family Caliciviridae [3]. The NoV genome is 7.5–7.7 kb in length and contains 3 open reading frames (ORFs). ORF1 encodes non-structural mature proteins (NS) that are involved in viral replication. ORF2 encodes the major structural protein (VP1), while ORF3 encodes a minor structural protein (VP2). NoVs are classified into seven distinct genogroups (G) I to VII on the basis of VP1 sequencing analysis [3]. NoV GI, GII, and GIV are known to infect humans; these genogroups can be subdivided into at least 33 genotypes [3, 4]. Among them, NoV GII is the most frequently detected in approximately 90% of NoV outbreaks, whereas NoV GI is associated with approximately 10% of NoV outbreaks, while NoV GIV is rarely detected [5]. Recently, a cell culture system for human NoVs was reported but its efficacy has not fully been determined [6]. Since there are no efficient cell culture systems to propagate NoVs, technical advances in the field of NoV detection have mainly been accomplished with conventional

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reverse-transcription (RT)-PCR and enzyme immunoassays (EIAs) [1, 3, 7]. However, conventional RT-PCR assays are time-consuming and cumbersome because additional nested PCR steps are required due to the low assay sensitivity and specificity, and carry-over contamination is frequently detected [8]. Compared to the conventional RTPCR assays, the commercially available enzyme immunoassays (EIAs) method is an attractive alternative because it is rapid, inexpensive and technically easy [1]. However, EIAs are generally not sensitive enough to detect a wide range of NoVs [1, 3]. During the first decade of the ­21st century, the introduction of real-time RT-quantitative PCR (RT-qPCR) assays substantially contributed to advances in NoV detection [3, 9]. This is since their use of target-specific DNA probes provides a more straightforward, sensitive and specific diagnostic method when compared to conventional RT-PCR assays [9–11]. The primary transmission route of human NoVs is via fecal-to-oral spread, although NoVs can disseminate through ingestion of contaminated water or food, exposure to infectious vomitus droplets, or close contact with infected individuals [1]. In the USA, NoVs have been identified in over 58% of samples in which foodborne outbreaks of illness were associated with an etiological agent [12]. In addition, waterborne NoV-associated AGE outbreaks have been frequently reported worldwide [1, 13–15]. Many groups have adopted RT-qPCR assays to detect NoVs in food or water samples, as well as in clinical samples; however, the frequency of NoV detection in these cases is low due to the extremely low viral loads and the presence of PCR inhibitors in the samples [16–19]. The prototype RT-qPCR assay for NoVs was invented by Kageyama et al. [9]. However, Jothikumar et al. reported that the prototype RT-qPCR assay for NoV GI strains is less sensitive than the assay for NoV GII strains [11]. Thus, they developed an improved RT-qPCR assay that was more

efficient in detection of NoV GI strains, and successfully applied this assay to shellfish samples [11]. To further enhance the performance of NoV GI detection, we redesigned a TaqMan probe based on the Jothikumar study and evaluated the RT-qPCR assay with archived clinical samples [11]. This modified assay provided broader strain reactivity than Jothikumar’s assay. Furthermore, it was more effective in water samples that contained NoV GI strains associated with groundwater-borne AGE outbreaks.

Materials and methods Clinical specimens for validation In South Korea, when waterborne and foodborne disease outbreaks are reported to local health centers, all cases are promptly and thoroughly investigated by technically trained public health officials according to the epidemiological investigation guidelines in order to identify causal agents and prevent the spread of disease. Gyeonggi province Institute of Health and Environment (GIHE) has official laboratories to diagnose waterborne and foodborne disease outbreaks in Gyeonggi, a highly populated area that houses 24% of the South Korean population. The samples were obtained from the Waterborne Virus Bank (Seoul, Korea). All NoV-positive human fecal specimens used in this study were collected from human patients with AGE outbreaks in Gyeonggi between January 2006 and December 2012 [20]. To verify the novel RT-qPCR probe, JJV1PM, for NoV GI strains (Table 1), a total of 22 NoV-positive human fecal samples (including 10 NoV GI and 12 NoV GII genotypes from AGE outbreaks in Gyeonggi) were used to evaluate monoplex RT-qPCR assays (Table 2). A negative control group (n = 12) was tested to identify false-positive reactions with other enteric viruses or stool

Table 1  Oligonucleotides used for the semi-nested RT-PCR and RT-qPCR assays for detecting norovirus GI strains Genogroup I

Oligonucleotide

Sequences (5’–3’)

Polaritya

Locationb

Referencec

semi-nested RT-PCR

GI-F1M GI-R1M GI-F2 JJV1F JJV1R JJV1PT

CTG CCC GAA TTY GTA AAT GAT​GAT​ CCA ACC CAR CCA TTR TAC ATY TG ATG ATG​ATG​GCG TCT AAG GAC GC GCC ATG TTC CGI TGG ATG TCC TTA GAC GCC ATC​ATC​AT VIC-TGT​GGA​CAG​GAG​ATCG CAA​TCT​C-TAMRA VIC-TGT GGA CAG GAG ATC G-NFQ-MGB

+ + + +

5342 5671 5357 5282 5377 5319

[24] [24] [24] [11] [11] [11]

+

5319

this study

RT-qPCR

JJV1PM

TAMRA, 6-carboxytetramethylrhodamine; NFQ, non-fluorescent quencher; MGB, minor groove binder a

 +, forward primer: -, reverse primer

b c

 Nucleotide locations based on the Norwalk (GI) (accession no. M87661) sequences

 References that used the indicated primers and probes [11, 24]

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Detection of waterborne norovirus by qPCR Table 2  Validation of RT-qPCR assays with NoV strains and other enteric viruses

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NoV ­genotypea(strain)

GenBank No.

mean±standard deviation of Ct values DNA probe for NoV GI RT-qPCR

GI-1 (GG/0305/2010/KOR) GI-2 (GG/0230/2010/KOR) GI-3 (GG/0513/2011/KOR) GI-4 (GG/0227/2011/KOR) GI-5 (GG/1123/2007/KOR) GI-6 (GG/0326/2010/KOR) GI-7 (GG/0253/2011/KOR) GI-8 (GG/03128/2012/KOR) GI-9 (GG/0386/2010/KOR) GI-12 (GG/0235/2011/KOR) GII-2 (GG/12180/2012/KOR) GII-3 (GG/0127/2011/KOR) GII-4 (GG/02121/2011/KOR) GII-5 (GG/0643/2012/KOR) GII-6 (GG/04104/2011/KOR) GII-7 (GG/0107/2010/KOR) GII-8 (GG/0134/2009/KOR) GII-12 (GG/0164/2008/KOR) GII-13 (GG/0259/2008/KOR) GII-14 (GG/01170/2010/KOR) GII-16 (GG/0334/2008/KOR) GII-17 (GG/0229/2011/KOR) Group A Rotavirus Astrovirus Enteric Adenovirus Stool ­controle

KY427651 KY427652 KY427653 KY427654 KY427655 KY427656 KY427657 KY427658 KY427659 KY427660 KY427661 KY427662 KY427663 KY427664 KY427665 KY427666 KY427667 KY427668 KY427669 KY427670 KY427671 KY427672

JJV1PTb

JJV1PMc

38.7±1.2 38.5±1.2 38.6±0.9 37.6±0.9 35.4±0.4 37.9±0.6 35.1±0.8 -

34.4±0.7 34.0±0.6 32.0.±0.7 34.4±0.6 33.9±0.4 32.0±0.5 34.0±1.1 34.6±0.6 31.8±0.5 31.6±1.5 -

Difference of Ct ­valuesd

4.3±0.5 4.6±0.7 4.3±0.4 3.8±0.5 3.4±0.1 3.9±0.5 3.3±0.3 -

a

 NoV genotypes were determined according to reference [4]

b c

 DNA probes for NoVs used as a control, see reference [11]

 DNA probes for NoVs modified in this study

d e

 Calculated difference of Ct value; b-c

 Stool control means the fecal specimens that did not include NoV, group A rotavirus, astrovirus, or enteric adenovirus

components; other enteric viruses in human fecal samples including group A rotavirus (n = 3), astrovirus (n = 3) and enteric adenovirus (n = 3). Stool controls (n = 3) were fecal specimens which were collected from human patients however NoV, group A rotavirus, astrovirus and enteric adenovirus were not detected (Table 2). Group A rotavirus and enteric adenovirus antigens were detected from the stool supernatants using an enzyme-linked immunosorbent assay (ELISA) with BioTracer™ Group A Rotavirus kit and Adenovirus kit (Bio Focus Co., Uiwang, South Korea), respectively, according to the manufacturer’s instructions. Norovirus and astrovirus were detected by RT-PCR.

Water samples for application To test the performance of the modified RT-qPCR assay, a total of 327 water samples were collected between 2006 and 2012. These sample types consisted of 131 river-water samples that were the source of tap water, 155 groundwater samples that were used for drinking and food processing, and 41 tap water samples. Water sampling and concentration were conducted using standard procedures [21, 22]. Briefly, at least 200 liters of water were passed through positivelycharged 1-MDS filters (Zetapore; AMF-Cuno, Meriden, CT). After samples were collected, the filter was packaged using a sterile technique, placed at 4 °C, and transported to

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the division of health research at GIHE, where they were processed within 24 h. Filters were eluted twice with 1.0 liters of 0.05 M glycine buffer (pH 9.5) mixed with 1.5% beef extract. The eluted product was immediately adjusted to pH 3.5 with 1 N HCl. After 30 min of stirring, the concentrate was centrifuged at 7,000×g for 15 min. The pellets were re-suspended with 25 ml of 0.15 M sodium phosphate (pH 9.5) and stirred for 10 min at room temperature. The resuspended samples were centrifuged at 2,500×g for 10 min. The supernatants were collected and filtered through a disc filter of 0.22-μm pore size. The final concentrated samples were stored at −80°C for further analysis. RNA extraction The fecal specimens were diluted to 10% suspensions with phosphate-buffered saline (PBS), suspended and clarified by centrifugation at 2,000×g for 15 min. In order to detect NoVs with conventional RT-PCR or RT-qPCR assays, viral RNA was extracted from fecal supernatants and concentrated water samples using a QIAamp viral RNA mini-kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. Reverse transcription (RT)‑PCR for NoV genotyping To identify the genotype of NoVs from fecal and water samples, semi-nested RT-PCR targeting Region C was performed (Table 1) [23, 24]. The primer set for NoV GI, GIF1M and GI-R1M, amplified a 329-bp PCR product in a first-step RT-PCR. For fecal specimens, one-step RT-PCR was performed using an AccuPower Rocketscript RT-PCR kit (Bioneer, Daejeon, Korea) as follows: 40 min of reverse transcription (RT) at 48 °C; 3 min of denaturation at 94 °C; 35 cycles consisting of denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, and extension at 72 °C for 45 s; and then a final extension at 72 °C for 7 min. For water samples, the RT reaction used Superscript III (Invitrogen) and ­1st PCR reverse primer. cDNA was synthesized as follows: 40 min of reverse transcription (RT) at 48 °C; 15 min of inactivation at 94 °C. First PCR was conducted using an AccuPower PCR kit (Bioneer, Daejeon, Korea) as follows; 3 min of denaturation at 94 °C, 35 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, and extension at 72 °C for 45 s; and a final extension at 72 °C for 7 min. For semi-nested PCR amplification of the ­1st round PCR product of fecal and water samples to detect NoV GI, the GI-F2 forward and GI-R1M reverse primers were used with an AccuPower PCR kit (Bioneer, Daejeon, Korea). Initial denaturation was carried out at 94 °C for 3 min. This was followed by 25 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 45 s, with a final extension at 72 °C for 7 min. This

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yielded 314-bp long final PCR products for GI, which were further characterized by sequencing to confirm genotype. Nucleotide sequencing and phylogenetic analysis Final PCR products from fecal specimens were purified and bi-directionally sequenced with nested PCR primers for genotyping. However, the amplified fragments from water samples were purified and then cloned into pGEM-T Easy vector (Promega, USA) according to the manufacturer’s recommendations. Plasmids were purified and then sequenced. To determine the genotype of NoVs, phylogenetic analysis was conducted with reference strains using neighbor-joining analysis and bootstrap analysis (n = 1000; MEGA version 6.0 software; Tempe, AZ, USA) [4]. In order to compare with the validation results using human fecal samples, the genotypes of waterborne NoVs strain were also determined. The nucleotide sequences of clinical and waterborne NoVs were submitted to the GenBank database with the following accession numbers: KY419205–KY419207 and KY427646–KY427672. RT‑PCR for probe region The RT-qPCR probe used by Jothikumar et al. [11], hereafter referred to as the JJV1PT-probe, was modified in order to increase the detection efficiency for NoV GI strains. For the sequence of the probe-target region, the RT reaction used Superscript III (Invitrogen) and the PCR reverse primer, GIR1M. cDNA was synthesized as follows: 40 min of reverse transcription (RT) at 48 °C; 15 min of inactivation at 94 °C. PCR was conducted using JJV1F, the GI-R1M primer set and an AccuPower PCR kit (Bioneer, Daejeon, Korea) as follows; 3 min of denaturation at 94 °C, 35 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, and extension at 72 °C for 45 s; and a final extension at 72 °C for 7 min (Table 1). The amplified PCR fragments of 389 bp were purified and then cloned into the pGEM-T Easy vector (Promega, USA) according to the manufacturer’s recommendations. Plasmids were purified and then sequenced. Monoplex one‑step RT‑qPCR The same forward and reverse primers for RT-qPCR assays were used as described in the previous study in order to discriminate the effect the probe modification (Table  1) [11]. For one-step RT-qPCR assay, AgPath-ID One-Step RT-PCR Reagent (Ambion, Austin, TX, USA) was used in a Real-Time PCR System 7500 (Applied Biosystems, Forster City, USA). The 25-μl reaction mixtures contained 5.0 μl of template RNA, 12.5 μl of 2× RT-PCR buffer, 1 μl of 25× RT-PCR enzyme mix, 1.67 μl of detection enhancer, each primers (0.25 μM) and genogroup-specific TaqMan probe

Detection of waterborne norovirus by qPCR

(0.2 μM). The one-step RT-qPCR conditions consisted of RT for 30 min at 55 °C, followed by activation of Taq polymerase at 95 °C for 15 min, amplification for 45 cycles: denaturation at 95 °C for 15 sec and annealing-extension at 55 °C for 1 min as reported [11]. All tests of monoplex one-step RT-qPCR were conducted in triplicate, and all data obtained were analyzed within a threshold of 40 for the crossing threshold (Ct) value.

Results Modification of the RT‑qPCR probe for Norovirus GI strains To gain accurate sequence information for the probe-target region (the ORF1-ORF2 junction region; Region B) in NoV GI strains, 10 nucleotide (nt) sequences reflecting the probe-target region were calculated from archived 10 NoV GI genotypes; NoV GI.1, GI.2, GI.3, GI.4, GI.5, GI.6, GI.7, GI.8, GI.9 and GI.12 (Fig. 1). The sequence alignment was conducted to identify the nucleotide differences between RT-qPCR probes and the target regions of the NoV GI strains. The alignment analysis revealed that 7 strains of NoV GI genotype, GI.1, GI.2, GI.4, GI.5, GI.6, GI.7 and GI.9, had perfect matches between the target region and the two probes, JJV1PT and JJV1PM (Fig. 1). However, NoV GI.3, GI.8 and GI.12 strains had two nucleotide mismatches compared with the old 23-nt JJV1PT probe (Fig. 1). Thus, a shortened 16-nt probe, JJV1PM was made to minimize the nucleotide mismatches (Fig. 1). The JJV1PM probe had a single nucleotide polymorphism (SNP) within the probe-target region for GI.3, GI.8 and GI.12 strains (Fig. 1). In addition, to stabilize the hybridization between the shorter DNA probe and the target template, a minor groove binder (MGB, dihydrocyclo-pyrroloindole tripeptide, which increases the melting temperature with shorter oligonucleotides) was attached to the 3´-terminus of the JJV1PM (Table 1) [25] .

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Validation of RT‑qPCR assays with clinical samples The modified RT-qPCR assay (hereafter referred to as the JJV1PM-probe assay) was specific for NoV GI strains as well as the original JJV1PT-probe assay (Table  2). The validation assay was performed with 10 NoV GI strains, 12 NoV GII strains, 3 group A rotavirus samples, 3 astrovirus samples, 3 enteric adenovirus samples and 3 stool samples. No cross-reactivity with NoV GII strains or other enteric viruses was observed in the RT-qPCR assays (Table 2). However, all NoV GI strains in the validation panel were detected using the JJV1PM-probe assay, whereas NoV GI.3, GI.8 and GI.12 genotype strains were not detected with the JJV1PT-probe assay (Table 2). Furthermore, the Ct values of JJV1PM-probe assays for NoV GI.1, GI.2, GI.4, GI.5, GI.6, GI.7 and GI.9 strains were lower, by at least 3.3, than those in the JJV1PT-probe assay (Table 2). Application of the improved RT‑qPCR assay to detect waterborne‑NoV GI strains NoVs-associated AGE outbreaks are often caused by contaminated water [14]. Due to its increased strain reactivity, we hypothesized that the JJV1PM-probe assay could be used to detect NoVs in water samples. Within a threshold of 40 for the crossing threshold (Ct) value, we detected NoV GI strains in 13/327 water samples by the JJV1PM-probe assay, while the JJV1PT-probe assay indicated only 2/327 samples were positive (Table 3). Waterborne NoV GI strains were not detected in any of 41 tap-water samples, but were found in 5 groundwater and 8 river water samples using the JJV1PMprobe assay (Table 3). Among them, 4 groundwater borne NoV GI strains have been associated with three previous AGE outbreaks (Table 3). Genotypic analysis showed that 7 genotypes of NoV GI strains were detected using the improved JJV1PMprobe assay, whereas only 2 genotypes were detected using the JJV1PT-probe assay (Table  3). NoV GI.2 and GI.3

Fig. 1  Sequence alignment for the region targeted by the RTqPCR probes used for detecting NoV GI strains. Nucleotide locations are based on Norwalk GI (Accession no. M87661) sequence numbering. Nucleotide mismatches in individual NoV GI strains are denoted by bold characters, when compared to the RT-qPCR probes

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3394 Table 3  Comparison of the detected Ct values from waterborne samples containing NoV GI genotypes, using RT-qPCR assays

H.-G. Cho et al. Sample No.

2010-3 2006-11 2006-16 N2010-30 N2011-50 N2011-51 2008-2 N2008-1 2006-06 2006-23 2007-39 2011-12 N2012-32

Water type

Riverwater Riverwater Riverwater Groundwater Groundwater Groundwater Riverwater Groundwater Riverwater Riverwater Riverwater Riverwater Groundwater

GenBank no.

KY419205 KY419206 KY419207 KY427646 KC413405 KC413379 KY427647 KC413381 KY427648 KY427649 KY427650 -

NoV genotype

TaqMan probe(mean Ct) JJV1PT

JJV1PM

GI-1 GI-2

35.7±0.7 34.9±0.6 -

38.9±1.0 31.8±0.4 33.2±0.6 36.1±0.7 33.4±0.5 33.5±0.6 31.6±0.3 33.0±0.6 36.5±0.8 38.0±1.0 33.1±0.4 33.2±0.5 35.6±0.8

GI-3 GI-4 GI-5 GI-6 GI-10 untypeda

NoV outbreak

Cb Bb Bb Ab -

a

 Untyped denotes NoV GI positive by RT-qPCR, but that NoV GI genotypes were not determined by seminested RT-PCR

b

 Groundwater-borne NoV outbreaks reported from South Korea [20, 23]

strains from water samples were efficiently detected by the JJV1PM-probe assay, but neither 2 of 3 NoV GI.2 strains nor the two NoV GI.3 strains were detected by the JJV1PT-probe assay (Table 3). In addition, other NoV GI.1, GI.5, GI.6 and GI.10 strains were also detected with the JJV1PM-probe assay, but not with the JJV1PT-probe assay. NoV GI.2 and GI.4 strains were detected with the JJV1PT-probe assay but the associated Ct values were approximately 4 (GI.2) and 2 (GI.4) higher than JJV1PM-probe assay (Table 3).

Discussion Rapid laboratory confirmation is essential to help implementation of proper control measures to reduce the magnitude of NoV outbreaks [1]. Waterborne NoVs-associated outbreaks have often been associated with NoV GI strains [15]. Since there are no efficient cell culture systems to propagate NoVs, RT-qPCR assay is regarded as the standard detection method for food and water samples [3]. We modified a RT-qPCR assay in order to detect NoV GI strains, and successfully applied it in water samples. The improved assay detected a broader range of NoV GI genotypes than the previous RTqPCR assay. The JJV1PT-probe assay was developed approximately ten years ago and is included in the standard protocol of U.S. EPA Method 1615 [11, 26]. A comparison of four RT-qPCR assays reported that the previously developed JJV1PT-probe assay had the best performance for NoV GI strain detection [10]. The JJV1PT-probe assay and the new JJV1PM-probe assay had high specificity; as well as no cross-reactivity with NoV GII strains or other enteric viruses. However several

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NoV GI genotypes were not detected during the validation process using the JJV1PT-probe assay, both in the validation panel and during application with water samples. These results showed that JJV1PT-probe assay had narrower reactivity for NoV GI genotypes than our new JJV1PM-probe assay. Consistent with our results, a recent study revealed that the JJV1PT-probe assay for NoV GI showed the lowest performance compared to 3 other recent methods [26]. To date, a standardized RT-qPCR assay for NoV GI using the NV1Cpr-probe has been available for food and water samples (ISO/TS15216-1) [27]. In addition, a recent comparative study reported that a new RT-qPCR assay using a NIFG1P-probe showed higher performance than the NV1Cpr-probe RT-qPCR assay for NoV GI strains [26, 28]. However, an additional comparison study between the JJV1PM-probe assay and two newer assays, NV1Cpr- and NIFG1P-probe, was not performed in this study. Compared to JJV1PM-probe, the NV1Cpr- and NIFG1P-probes target the same ORF1-ORF2 junction (region B) but have: different lengths, NV1Cpr probe-20 nt and NIFG1P probe-22nt; 2 degenerative nucleotides in the middle of each probe; and different quencher dyes at the 3’-terminus [27, 28]. Without direct evaluation tests, we cannot conclude whether the JJV1PM-probe assay is more advantageous or disadvantageous than the two newer assays because they used different primer sets, RT-PCR conditions, quencher dyes and degenerative sequences within the probe. Therefore, further study is needed to explore the most efficient probe for RT-qPCR based diagnostics of NoV GI strains. A major impediment to detecting NoVs using PCR-based methods is the high genomic diversity of these pathogens, which makes it difficult to design primers and probes that

Detection of waterborne norovirus by qPCR

can detect all NoV genotypes with equal efficiency [7, 9]. The ORF1-ORF2 junction (region B) is the most appropriate site for primer/probe design because it is the most conserved region of the NoV genome [1, 9]. However, new variant NoV strains may have critical mismatches in these probe-targeted regions due to the genomic diversity of NoVs generated by genetic drift [29]. A recent study reported that a single nucleotide polymorphism (SNP) in the TaqMan probe-binding region reduced the strain reactivity of a RT-qPCR assay for detection of NoV GII.4 Sydney strains [30]. Consistent with this, we found that NoV GI strains, in which the probetargeted region had more than one mismatch, could not be detected efficiently with the JJVIPT-probe assay. The probetargeted regions of NoV GI.3, GI.8, and GI.12 strains used in our validation panel had double-nucleotide mismatches with the JJVIPT-probe, whereas they had a SNP and were detected by the JJV1PM-probe. This suggests that the number of nucleotide mismatches in the probe-targeted region of NoVs might influence the strain reactivity of the RT-qPCR assay through weak probe-template hybridization [30]. Although the target sequences of several NoV GI genotypes were identical between the JJV1PT- and JJV1PMprobes, they were detected with higher Ct values by the JJV1PT-probe assay in our validation panels. In addition, the JJV1PT-probe assay detected just two NoV GI strains in water samples, whereas the JJV1PM-probe assay detected various NoV GI genotypes, albeit in the context of high Ct values. These results indicate that the JJV1PM-probe assay had lower Ct-values as well as broader strain reactivity in water and clinical samples than the JJV1PT-probe assay. Under the same assay conditions, the JJV1PM-probe was conjugated to a different quencher dye (NFQ, non-fluorescent quencher) and had a minor groove binder (MGB) at its 3´ end. Nucleotide probes with MGBs are known to efficiently quench reporter dyes through their close proximity; they also efficiently stabilize hybridization between the DNA probe and the target template [25]. NFQ also has an improved signal to noise (S/N) ratio compared to the fluorescent quencher, TAMRA [31]. These results suggest that the effect of NFQ and/or MGB at the 3´ end of JJV1PM-probe contributes to the lower Ct-values and broader strain reactivity of our JJV1PM-probe assays, as previously reported [31, 32]. The optimal RT-qPCR assay for NoV detection should have high sensitivity as well as high specificity and broad strain reactivity [3, 26]. The new JJV1PM-probe assay showed high specificity and broader strain reactivity throughout the validation and application tests. However, the conclusive experimental data for assay-sensitivity, PCR-amplification efficiency and detection limit tests using diluted target-RNA transcripts, are not presented in this study, even though the JJV1PM-probe assay showed lower Ct-values than JJV1PT-probe assay. In addition, the

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probe-target region sequences from the NoV GI strains detected in the water samples should have been aligned to show separate data to re-confirm the broader strain reactivity of the JJV1PM-probe based assay. However, NoV sequences from water samples were not obtained from RT-PCR assays presumably owing to the limitation of our single-round, rather than nested, PCR method. These aspects mentioned above are limitations of this study, which should be cautiously interpreted. In conclusion, the validation of RT-qPCR assays is needed with recently circulating NoV strains prior to field application because of the ongoing emergence of new variants and the high genetic diversity of NoVs. The new JJV1PM-probe assay had high specificity and broader strain reactivity through our validation tests and and was successfully applied to the detection of waterborne NoV GI strains. As NoV GI strains are an important etiological agent of waterborne or foodborne AGE outbreaks, the improved RT-qPCR assay will be of great utility to public health research groups to prevent further NoV GI-associated AGE outbreaks. Acknowledgements  This research was supported by a Grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant numbers: HI15C1781). Compliance with ethical standards  Conflict of interest  The authors have declared that no competing interests exist. Ethical approval  This article does not contain any studies with human participants performed by any of the authors.

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