Research Article Development of Enhanced Primer Sets for

Research ArticleDevelopment of Enhanced Primer Sets forDetection of Norovirus

Byoung-Hwa Kong1 Sung-Geun Lee2 Sang-Ha Han1 Ji-Young Jin1

Weon-Hwa Jheong3 and Soon-Young Paik1

1Department of Biomedical Sciences College of Medicine The Catholic University of Korea 222 Banpo-daeroSeocho-gu Seoul 137-701 Republic of Korea2Korea Zoonosis Research Institute Chonbuk National University Iksan 570-390 Republic of Korea3Environmental Infrastructure Research Department National Institute of Environmental ResearchIncheon 404-708 Republic of Korea

Correspondence should be addressed to Soon-Young Paik paikcatholicackr

Received 29 August 2014 Revised 5 November 2014 Accepted 27 November 2014

Academic Editor Elena Orlova

Copyright copy 2015 Byoung-Hwa Kong et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Norovirus (NV) is a major viral pathogen that causes nonbacterial acute gastroenteritis and outbreaks of food-borne disease Thegenotype of NV most frequently responsible for NV outbreaks is GII4 which accounts for 60ndash80 of cases Moreover originaland new NV variant types have been continuously emerging and their emergence is related to the recent global increase in NVinfection In this study we developed advanced primer sets (NKI-FRF2 NKII-FRR2) for the detection of NV including thevariant types The new primer sets were compared with conventional primer sets (GI-F1R1F2 SRI-123 GII-F1R1F2 and SRII-123) to evaluate their efficiency when using clinical and environmental samples Using reverse transcription polymerase chainreaction (RT-PCR) and seminested PCR NV GI and GII were detected in 917 (NKI-FRF2) 893 (NKII-FRR2) 542 (GI-F1R1F2) 525 (GII-F1R1F2) 250 (SRI-123) and 322 (SRII-123) of clinical and environmental specimensTherefore ourprimer sets perform better than conventional primer sets in the detection of emerged types of NV and could be used in the futurefor epidemiological diagnosis of infection with the virus

1 Introduction

Norovirus (NV) belonging to family Caliciviridae is a majorcause of acute viral gastroenteritis [1] Although symptomswhich typically appear between 12 and 48 h are generallymild and self-limiting they can be severe in immunocom-promised groups such as infants and the elderly [2 3]Viral infection is primarily related to foodborne illness butperson-to-person contact and waterborne outbreaks are alsoimportant vehicles for transmission [4ndash7]

The NV genome is composed of approximately 77 kbof single stranded positive sense RNA (+ssRNA) whichincludes three open reading frames (ORFs) ORF1 ORF2and ORF3 [8] Six nonstructural proteins in a polyprotein areencoded by ORF1 including an RNA-dependent RNA poly-merase (RdRp) [9] ORF2 and ORF3 encode major structuralcapsid protein (VP1) and minor structural capsid protein

(VP2) respectively [10] VP1 consists of a shell domain (S)and two protruding (P) domains [11] The P1 domain a pro-truding flexible hinge region is located between the S and P2domains [12] The P2 domain is a hypervariable region thatbinds to host cell [13]The stability of VP1 is increased byVP2which prevents its degradation [14]

NV is classified into six groups genogroups I to VI(GI to GVI) based on the amino acid sequences of theRdRp and VP1 [5 15 16] The genogroups GI GII andGIV are found in humans [5] Outbreaks appear more fre-quently in GI GII than GIV [17ndash20] In particular GII4 hasemerged continuously every 2-3 years in an evolved form [21]Consequently it accounts for 87 of the NV outbreaks thatoccur globally [22ndash24]

In the Republic of Korea NV GII4 Sydney type emergedbetween 2012 and 2013 during which time it accounted for604 of NV GII4 diagnoses [25] Detection of the NV GII

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 103052 9 pageshttpdxdoiorg1011552015103052

2 BioMed Research International

Table 1 Description of NV GI sequences for genetic analysis

Accessionnumber(GenBank)

Genotype StrainAccessionnumber

(GenBank)Genotype Strain

EU085529 GI1 P774Delsjo2004GothenburgSweden KF039731 GI1 CH4XO5332009USA

FJ515294 GI2 Leuven2003BEL KF039732 GI1 CHA5A0102009USA

JN176918 GI2 Roosendaal0292006NL KF039733 GI1 CHA9A004 201104262011USA

JN183159 GI9 S482008Lilla KF039734 GI1 CHA6A0142009USA

JN183161 GI7 S242008Lilla Edet KF039735 GI1 CHA3A0072008USA

JN603244 GI3 S292008Lilla EdetSweden KF039736 GI1 CHA7A0112010USA

JN603245 GI4 S502008Lilla EdetSweden KF039737 GI1 CHA6A003 200911042009USA

JQ388274 GI6 KingstonACT160D2010AU KF306212 GI2 Jingzhou2013401CHN

JQ743331 GI4 2000 KF429761 GI1 8MoIIIL1972USA

JQ743332 GI2 1999 KF429765 GI1 8W1968USA

JQ911594 GI 103602010VNM KF429770 GI1 8McIII1973USA

JX023285 GI1 8FIIa1968USA KF429773 GI1 8CKIIIc1974USA

KC998959 GI6 TCH-099USA2003 KF429774 GI1 8UIIIf1973USA

KF039725 GI1 CHA7A0092010USA KF429783 GI1 8K1979USA

KF039726 GI1 CHA6A003 200910312009USA KF429789 GI1 8MC1978USA

KF039727 GI1 CHA2A0142008USA KF586507 GI9 CAIQ12110628

KF039728 GI1 CHA2A0142008USA L07418KF039729 GI1 CHA6A0072010USA M87661KF039730 GI1 CHA9A004 201104192011USA

strain is difficult with existing RT-PCR primer sets (GII-F1R1F2 SRII-123) because of the continuous variation ofthe strain [26 27] In addition GI-F1R1 primer set does notalways have sufficient specificity to detect NV because false-positive detection commonly occurs [28]

Therefore the aim of this studywas to develop primer setsfor efficiently detectingNVGI andGII including detection ofnewly emerged strains that could not previously be identifiedwith conventional primer sets Once new primer sets weredeveloped we evaluated their efficiency using an RT-PCRassay to test clinical and environmental specimens

2 Materials and Methods

21 Collection of Clinical and Environmental Samples Twosample types clinical and environmental specimens wereused for detection of NV GI and GII Eighty-six unknownsamples were used for detection of NV GI They included22 clinical samples from Gyeonggi Institute of Health Envi-ronment (GIHE) that were originally obtained during 2012and 2013 and 24 clinical and 40 environmental samples fromWaterborne Virus Bank (WAVA) originally obtained from2006 to 2013 To identify NVGII we used 134 unknown sam-ples that included 35 clinical samples from GIHE originallycollected during 2012 and 2013 and 32 clinical and 67 envi-ronmental samples fromWAVA collected from 2006 to 2013

All stool specimens were collected from patients whosuffered from diarrhea caused by acute gastroenteritis Envi-ronmental specimenswere collected fromgroundwater in theRepublic of Korea during June and October 2013 All sampleswere stored at minus80∘C until use

22 Ethical Clearance All clinical samples were obtainedduring themedical treatment of patients with acute gastroen-teritis All patients providedwritten informed consent whichhas been kept on file at the GIHE and WAVA Human rightswere not abused nor were ethical issues encountered duringthe study All experimental work and collection of sampleswere supervised and approved by the Institutional ReviewBoard (IRB) of SongeuiMedical CampusTheCatholic Univ-ersity of Korea (approval number MC14SISI0039)

23 Primer Design In order to design new primer sets37 sequences of NV GI (Table 1) and 52 sequences of NVGII (Table 2) were obtained from NCBI and imported intoEditSeq and MegAlign in DNASTAR software (DNASTARUSA)

We designed 3 candidates for the NV GI primer set and 5candidates for theNVGII primer set in the conserved regionsof ORF 1 and ORF 2 Among them NKI and NKII (Table 3)which have outstanding efficiency were selected Inner pri-mers were designed to detect NV fromwater samples because

BioMed Research International 3

Table 2 Description of NV GII sequences for genetic analysis




AB447433 GII4 Aormori2006JP JQ320072 GII2 NF2002USA2002AB541319 GII4 Osaka2007JP JX445152 GII4 AlbertaE1312004CAAB541321 GII4 Osaka22007JP JX445153 GII4 AlbertaEI1422006CAAB541362 GII4 Toyama52008JP JX445157 GII4 AlbertaEI5132006CAAB543808 GII4 FUMI2010JP JX445159 GII4 AlbertaEI0092008CAAB662873 GII-2 OC091042009JP JX445161 GII4 AlbertaEI2102008CAAY485642 GII4 Langen 10612002GER JX445168 GII4 AlbertaEI3882008CAAY502023 GII4 Farmington Hills2002USA JX459900 GII4 NSW882J2011AUDQ658413 GII4 MD-20042004USA JN899245 GII21 Salisbury1502011USADQ078814 GII4 Hunter504D2004AU JX459907 GII4 NSW33092012AUEF202588 GII4 Toronto SK2005CAN JX459908 GII4 NSW05142012AUEU310927 GII4 TCH186 2002 US JX846924 GII3 HK711978CHNEF684915 GII4 Shellharbour NSW696T2006AUS JX846925 GII2 KL1091978MYSFJ537135 GII4 CHDC20941974US JX846926 GII7 CHDC39361988USAGQ645366 GII4 NSW36392008AUS JX989075 GII6 GZ2010-L96GuangzhouCHN 2011GQ845368 GII4 NSW505G 2007 AUS KC175323 GII4 Hong kong CUHK36302012CHNGQ845369 GII4 Armidale NSW39012008AU KC464499 GII12 CGMH412010TWGU017907 GII14 8594Maizuru2008 JPN KC464505 GII2 CGMH472011TWGU134965 GII7 17382009USA KC576910 GII6 S9c1976SENGU445325 GII4 New Orleans18052009USA KC597138 GII2 CHDC25961975USAGU969058 GII13 8679Maizuru2008 JPN KC597139 GII17 C1421978GUFGU980585 GII3 CBNU12006KOR KC597140 GII3 NIHIC812011USAHQ664990 GII12 HS2062010USA KF306213 GII3 Jingzhou2013402CHNJN400623 GII4 CGMH252010TW KF429764 GII1 21K11972USAJN595867 GII4 Ascension2082010 KF429769 GII2 SnowMountRS1975USAJN797508 GII1 HawaiiTD1974USA X86557 GII4 Lordsdale virus

NVgenerally presentswith a low titer inwater [29]Theywereconstructed based on conserved sites fromprimer sets NKI-FandNKII-RWe named the inner primers NKI-F2 andNKII-R2 and used them for nested PCR

24 Viral RNA Extraction Viral RNA was extracted fromclinical and environmental specimens with the QIAampviral RNA extraction kit (Qiagen Germany) according tothe manufacturerrsquos instructions Each viral RNA sample waseluted with 60 120583L of elution buffer and stored at minus80∘C untiluse in the RT-PCR assay

25 RT-PCRand Seminested PCR ExtractedRNAwas ampli-fied by both RT-PCR and seminested PCR in S1000 ThermalCycler (BIO-RAD Singapore) In RT-PCR extracted RNAwas reverse-transcribed and amplified using the QIAGENOneStep RT-PCR Kit (Qiagen Germany) according to themanufacturerrsquos protocolThe total reactionmixture volumeof25 120583L contained the following 5timesQIAGENOneStep RT-PCRbuffer (5 120583L) 20 pmol primers (1 120583L each) (NKI-FR NKII-FR SRI-12 SRII-12 GI-F1R1 and GII-F1R1) 10mMdNTPmix (1120583L) enzymemix (reverse transcriptase and Taq

polymerase 1 120583L) extracted viral RNA template (3120583L) andRNase-free water (13 120583L) (Welgene Republic of Korea) RT-PCR conditions were as follows reverse transcription at 50∘Cfor 30min initial PCR activation at 95∘C for 15min 39 cyclesof denaturation at 94∘C for 30 s annealing at each optimalannealing temperature (Tables 4 and 5) for 30 s extension at72∘C for 1min and a final extension at 72∘C for 10min

For seminested PCR the RT-PCR product (3 120583L)20 pmol primers (1 120583L each) (NKI-F2R NKII-FR2 SRI-23SRII-23 GI-F2R1 and GII-F2R1) and RNase-free water(15 120583L) were added to a Maxime PCR PreMix Kit (119905-StarTaq)(iNtRONBiotechnology Republic of Korea) PCR conditionswere as follows initial denaturation at 94∘C for 2min 20cycles of denaturation at 94∘C for 20 s annealing at each opti-mal annealing temperature (Tables 4 and 5) for 10 s extensionat 72∘C for 20 s and a final extension at 72∘C for 5min

26 Sequence Analysis of PCR Products The products of RT-PCR and seminested PCRwere analyzed using 2 agarose gelelectrophoresis in TAE buffer with DNA SafeStain solution(Lamda Biotech USA) and were purified in agarose gel witha HiYield GelPCR DNA Fragments Extraction Kit (RBC


Table 3 The nucleotide sequences of NV GI (a) and NV GII (b) strains were compared with sequences of designed primer sets

(a) GI

NKI primer set

Primer NKI-F (forward)G T A A A T G A T G A T G G C G T C T A A

M87661 (GI) 0KF039732 (GI1) 0KF306212 (GI2) 0JN603244 (GI3) 0JN603245 (GI4) 0JQ388274 (GI6) 0JN183161 (GI7) 0KF586507 (GI9) 0

Primer NKI-R (reverse)A C C C A D C C A T T R T A C A T Y T G

M87661 (GI) 0KF039732 (GI1) 0KF306212 (GI2) 0JN603244 (GI3) G 1JN603245 (GI4) 0JQ388274 (GI6) 0JN183161 (GI7) 0KF586507 (GI9) 0

(b) GII

NKII primer set

Primer NKII-F (forward)C T Y A G G C A R A T G T A C T G G A C Y

X86557 (GII) 0JN797508 (GII1) A T 2KF429769 (GII2) 0KF306213 (GII3) A T 2JX459907 (GII4) 0KC464499 (GII12) A T 2KC597139 (GII17) 0JN899245 (GII21) A 1

Primer NKII-R (reverse)T C G A C G C C A T C T T C A T T C A C

X86557 (GII) 0JN797508 (GII1) 0KF429769 (GII2) 0KF306213 (GII3) 0JX459907 (GII4) 0KC464499 (GII12) 0KC597139 (GII17) 0JN899245 (GII21) 0


Table 4 Information of NV GI primer sets

Primers Sequence (51015840-31015840)a Locationb Region Annealing temperature (∘C) Polarity ReferenceNKI-F GTA AAT GAT GAT GGC GTC TAA 5354ndash5373

Capsid 51+

This studyNKI-R ACC CAD CCA TTR TAC ATY TG 5649ndash5668 minus

NKI-F2 GAT GGC GTC TAA GGA CGC 5363ndash5380 +GI-F1 CTG CCC GAA TTY GTA AAT GAT GAT 5342ndash5365

Capsid 55+

[26]GI-R1 CCA ACC CAR CCA TTR TAC ATY TG 5649ndash5671 minus

GI-F2 ATG ATG ATG GCG TCT AAG GAC GC 5358ndash5380 +SRI-1 CCA ACC CAR CCA TTR TAC AT 5652ndash5671

Capsid 50minus

[27]SRI-2 AAA TGA TGA TGG CGT CTA 5356ndash5373 +SRI-3 AAA AYR TCA CCG GGK GTA T 5578ndash5596 minus

aThe means of alphabet sequence are the following Y = C T R = A G K = G T D = A G TbLocation is based on accession number M87661 (Norwalk virus)

Table 5 Information of NV GII primer sets

Primers Sequence (51015840-31015840)a Locationb Region Annealing temperature (∘C) Polarity ReferenceNKII-F CTY AGG CAR ATG TAC TGG ACY 4805ndash4825

RdRp 55+

This studyNKII-R TCG ACG CCA TCT TCA TTC AC 5081ndash5100 minus

NKII-R2 GGA GCC AGA TTG CGA TCG C 5060ndash5078 minus

GII-F1 GGG AGG GCG ATC GCA ATC T 5049ndash5067Capsid 55

+[26]GII-R1 CCR CCI GCA TRI CCR TTR TAC AT 5367ndash5389 minus

GII-F2 TTG TGA ATG AAG ATG GCG TCG ART 5079ndash5102 +SRII-1 CGC CAT CTT CAT TCA CAA A 5078ndash5096

RdRp 50minus

[27]SRII-2 TWC TCY TTY TAT GGT GAT GAT GA 4583ndash4605 +SRII-3 TTW CCA AAC CAA CCWGCT G 4767ndash4785 minus

aThe means of alphabet sequence are the following Y = C T R = A G I = inosine W = A TbLocation is based on accession number X86557 (Lordsdale virus)

Taiwan) For sequencing PCR products were sent to CosmoGenetech (Republic of Korea) and returned sequences wereanalyzed using the basic local alignment search tool (BLAST)in NCBI

27 Statistical Analysis An efficiency test of both GI primersets (GI-F1R1F2 SRI-123 and NKI-FRF2) and GIIprimer sets (GII-F1R1F2 SRII-123 andNKII-FRR2) wasstatistically analyzed using SPSS 200 [30]

28 Nucleotide Sequence Registration The NV GI and NVGII sequences that were submitted to GenBank in NCBI(httpwwwncbinlmnihgov) were isolated from stool andgroundwater samples

3 Results

31 Selection of Primer Sets Collected NV sequences (37from NV GI 52 from NV GII) were aligned using EditSeqand MegAlign in DNASTAR software Results showed thatconserved sequences were selected in ORF1 and ORF2

From the 3 and 5 respective candidate NVGI andNVGIIprimer sets that were evaluated for efficiency using clinicalsamples themost efficient primer sets were selected and usedin this study (Table 3)

32 Detection of NV GI and GII To evaluate the efficiencyof the new primer sets (NKI-FRF2 NKII-FRR2) designedfor this study we used RT-PCR and compared them toconventional primer sets for NV GI (GI-F1R1F2 SRI-123)and GII (GII-F1R1F2 SRII-123) The ORF1 region wasamplified by NKII-FRR2 and SRII-123 while the ORF2region was amplified by NKI-FRF2 SRI-123 GI-F1R1F2and GII-F1R1F2 (Figure 1)

ForNVGI 84 samples were confirmedwithGI-F1R1F2SRI-123 and NKI-FRF2 detecting 13 6 and 22 samplesrespectively For NV GII 134 samples were identified usingGII-F1R1F2 SRII-123 and NKII-FRR2 to detect 31 19and 53 samples respectively

33 Comparison of Primer Sets In total 83 positive sampleswere identified including 50 clinical (18 NV GI 32 NV GII)and 33 environmental (6 NV GI 27 NV GII) specimens Themost sensitive primer set for NV GI was NKI-FRF2 whichdetected 2224 positive samples (917) In comparison theother primers detected 1324 (542 GI-F1R1F2) and 624(250 SRI-123) samples containing NV GI

In detecting NV GII the NKII primer set showed a supe-rior diagnostic yield compared to the other sets NKIIFRR2identified 5359 (893) positive NV GII samples whereasGII-F1R1F2 and SRII-123 identified 3159 (525) and


3998400

5998400

RdRp

NKI-FR (5354ndash5666)SRI-12 (5356ndash5671)

GI-F1R1 (5342ndash5671)

ORF1 ORF2 ORF3

(a)

3998400

5998400

RdRp

NKII-FR (4805ndash5100)SRII-12 (4583ndash5096)

GII-F1R1 (5058ndash5401)

ORF1 ORF2 ORF3

(b)

Figure 1 Location of designed primer (NKI FR NKII FR) and conventional primers (SRI-12 SRII-12 GI-F1R1 and GII-F1R1) of NVGI (a) and GII (b)

Table 6 Sensitivity of NV GI using each primer set

Positive specimens NKI FRF2 GI F1R1F2 SRI 123GI3 3 3 mdashGI4 6 2 4GI6 5 4 1GI8 1 mdash 1GI15 1 mdash mdashGI 1 1 mdashClinical samples(119899 = 18) 17 (944)lowast 10 (588) 6 (333)

GI3 4 3 mdashGI6 1 mdash mdashEnvironmentalsamples (119899 = 6) 5 (833) 3 (500) 0 (0)

Total (119899 = 24) 22 (917) 13 (542) 6 (250)All 119875 values lt 005 are by 119905-test except for environmental samples (GIF1R1F2 and NKI FRF2)Environmental samples were not detected by SRI 123 primerlowastPercentage is NV detectionNV positive ratio

1959 (322) respectively Sensitivity using each primer setis shown in Table 6 (NV GI) and Table 7 (NV GII) SRI-123and SRII-123 were unsuitable for use with environmentalsamples because positive samples were not detected

34 Sequence Analysis Results of BLAST sequence analysisindicated that some of the PCRproducts were amplified usingseveral primers (GI-F1R1F2 GII-F1R1F2 and SRI-123)which were shown as false positives of NV whereas NKI-FRF2 NKII-FRR2 and SRII-123 did not detect falsepositives for NV However NKI-FRF2 and SRI-123 diddetect NVGII in some samplesWhere NVGII was detectedit had similar sequences which were as follows AB290150(NKI-FRF2) FJ383875 (NKI-FRF2 SRI-123) KF289337(SRI-123) and KF509946 (NKI-FRF2 SRI-123)

Genotypes of NV GI3 GI4 GI6 GI8 and GI15 andNVGII2 GII3 GII4 GII6 GII13 GII16 GII17 andGII21were identified using GI and GII primer sets respectively(Tables 6 and 7) Using all primer sets 17 samples containingthe NV GII4 variant were identified NKII-FRR2 SRII-123 andGII-F1MR1MF3MdetectedNVGII4 variant typein 16 12 and 10 samples respectively

From positive samples amplified sequences obtainedusing NKIFRF2 and NKII-FRR2 were registered in NCBIThe deposited accession numbers were as follows KJ742428KJ742429 KJ742430 KJ742431 KJ742432 KJ742433

Table 7 Sensitivity of NV GII using each primer set

Positive specimens NKII FRR2 GII F1R1F2 SRII 123GII2 2 1 1GII3 1 mdash 1GII4 18 11 12GII6 2 1GII13 mdash 1 mdashGII16 1 1 mdashGII17 4 2 4GII21 1 mdash mdashGII 1 1 1Clinical samples(119899 = 32) 30 (938)lowast 18 (563) 19 (594)

GII4 22 11 mdashGII 1 2 mdashEnvironmentalsamples (119899 = 27) 23 (857) 13 (481) 0 (0)

Total (119899 = 59) 53 (893) 31 (525) 19 (322)All 119875 values lt 005 are by 119905-testEnvironmental samples were not detected by SRII 123 primer setlowastPercentage is NV detectionNV positive ratio

KJ742434 KJ742435 KJ742436 KJ742437 KJ742438KJ742439 KJ742440 KM017944 KM017945 KM017946KM017947 KM017948 KM017949 KM017950 KM017951KM017952 KM017953 KM017954 KM017955 KM017956KM017957 KM017958 and KM017959

4 Discussion

In the USA estimated 570ndash800 people die annually fromoutcomes associated with NV Outbreaks of the virus occurregularly and new variant strains are identified worldwideevery 2 to 3 years [3 21 31 32] Methods for detectionof NV include RT-PCR the enzyme-linked immunosorbentassay (ELISA) and transmission electron microscopy (TEM)[33 34] In previous studies virus concentrations up to 102virus-copiesmL were detected using RT-PCR compared to105 virus-copiesmLwith othermethods [33]Thewidespreaduse of RT-PCR for NV detection can be attributed to itssuperior sensitivity compared to other methods [35 36] Forexample it is the recommended method for detection ofNV in contaminated water in which NV commonly existsat 491times 102ndash351 times103 copiesmL [29] However in order tominimize outbreaks and prevent potential deaths improved


primer sets are required to accurately detect original andvariant strains of NV

It is possible that the primer sets used for detection of NVare different in each laboratory because a standard methodfor detection has not yet been established [37] In previouslypublished work the primer sets GI-F1R1F2 and GII-F1R1F2 SRI-123 and SRII-123 have typically been usedto detect NV [38 39] Indeed GI-F1R1F2 and GII-F1R1F2are recommended for detection of NV by the Centers forDisease Control and Preventionrsquos (CDC) of Republic of Korea(httpwwwcdcgokrCDCcontentsCdcKrContentViewjspcid=18302ampmenuIds=HOME001-MNU1175MNU834-MNU0839) Lee et al (2011) used SRI-123 SRII-123 GI-F1R1F2 and GII-F1R1F2 primer sets when testing watersamples collected in 2008 near groundwater in Republicof Korea [40] They found that 117 sites were contaminatedwith NV GI and GII and the study indicated that NV couldmore efficiently be detected with GI-F1R1F2 (35 samples)and GII-F1R1F2 (55 samples) compared to SRI-123 (27samples) and SRII-123 (41 samples) [40] Similarly in thepresent study the performance of SRI-123 and SRII-123in detection of NV was inferior to other primer sets Inparticular SRI-123 and SRII-123 entirely failed to detectNV in water Conversely the majority of positive NV sampleswere detected using NKI-FRF2 (917) and NKII-FRR2(893) We postulate that because the sequences of NKI-FRF2 and NKII-FRR2 were collected from 1968 to 2013their design containedmore conserved sequences in compar-ison to the GI-F1R1F2 GII-F1R1F2 SRI and SRII primersets and this could explain their superior performance

NV GII4 is known to be the most prevalent strain of NV[41] and since 2012 a new variant of NV GII4 has causedmany cases of gastroenteritis [42] In detecting NV GII4 anRT-PCR assay using a GII-F1R1F2 primer set or anotherforward primer (Cog2F F2 FB GV21) with a GII-F1R1F2reverse primer has typically been used [25 42ndash44] Howeverthese primer sets were designed before 2005 and are notsuitable for detection of new NVmutant types that have con-tinuously emerged since such as the 2012 Sydney strainTheirunsuitability is probably explained by differences in sequencebetween the primers and the new variant types [45 46]

In this study NKII-FRR2 GII-F1R1F2 and SRII wereused to detect the NV GII4 variant In total 17 NV GIIvariant samples were confirmed using NKII-FRR2 SRII-123 and GII-F1R1F2 They detected 16 12 and 10 NV GIIsamples respectively The GII-F1R1F2 primer set has beenwidely used for detection of NV GII4 [42ndash44 47] Howeverin previous research the efficiency of this primer set fordetectingNVGII4 was relatively low [25 41]Therefore GII-F1R1F2may be an inappropriatemethod for detection of theNV GII4 variant

5 Conclusions

In our study we showed that the NKI-FRF2 and NKII-FRR2 primer sets could be important for epidemiologicaldiagnosis of NV in the laboratory because of their superiorperformance compared to other primer sets Where contam-ination with NV was suspected these primer sets could be

applied to specimens taken fromwater sample with a low titeror clinical samples with a high titer Additionally our newlydeveloped primer sets can be used to detect variant types ofNV Therefore we recommend the use of NKI-FRF2 andNKII-FRR2 in future research

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was supported by the National ResearchFoundation of Korea (NRF-2012R1A2A2A01045078)and Korea Environmental Industry amp Technology Institute(2013000550009)

References

[1] S Maritschnik E E Kanitz E Simons et al ldquoA food handler-associated foodborne norovirus GII4 Sydney 2012-outbreakfollowing a wedding dinner Austria October 2012rdquo Food andEnvironmental Virology vol 5 no 4 pp 220ndash225 2013

[2] S M Griffin N E Brinkman E J Hedrick E R Rhodes andG S Fout ldquoComparison of nucleic acid extraction and reversetranscription-qPCR approaches for detection of GI and GIInoroviruses in drinking waterrdquo Journal of Virological Methodsvol 199 pp 76ndash85 2014

[3] MM Patel A J Hall J Vinje andUD Parashar ldquoNorovirusesa comprehensive reviewrdquo Journal of Clinical Virology vol 44 no1 pp 1ndash8 2009

[4] K Mattison ldquoNorovirus as a foodborne disease hazardrdquoAdvances in Food and Nutrition Research vol 62 pp 1ndash39 2011

[5] D-P Zheng T Ando R L Fankhauser R S Beard R I Glassand S S Monroe ldquoNorovirus classification and proposed strainnomenclaturerdquo Virology vol 346 no 2 pp 312ndash323 2006

[6] S Svraka E Duizer H Vennema et al ldquoEtiological role ofviruses in outbreaks of acute gastroenteritis inThe Netherlandsfrom 1994 through 2005rdquo Journal of Clinical Microbiology vol45 no 5 pp 1389ndash1394 2007

[7] L Barclay G W Park E Vega et al ldquoInfection control fornorovirusrdquoClinicalMicrobiology and Infection vol 20 no 8 pp731ndash740 2014

[8] K Y Green T Ando M S Balayan et al ldquoTaxonomy of thecalicivirusesrdquo The Journal of Infectious Diseases vol 181 sup-plement 2 pp S322ndashS330 2000

[9] G Belliot S V Sosnovtsev T Mitra C Hammer M Garfieldand K Y Green ldquoIn vitro proteolytic processing of the MD145Norovirus ORF1 nonstructural polyprotein yields stable pre-cursors and products similar to those detected in calicivirus-infected cellsrdquo Journal of Virology vol 77 no 20 pp 10957ndash10974 2003

[10] M E Hardy andM K Estes ldquoCompletion of the Norwalk virusgenome sequencerdquoVirus Genes vol 12 no 3 pp 287ndash290 1996

[11] E F Donaldson L C Lindesmith A D Lobue and R S BaricldquoViral shape-shifting norovirus evasion of the human immunesystemrdquo Nature Reviews Microbiology vol 8 no 3 pp 231ndash2412010


[12] A Bertolotti-Ciarlet L J White R Chen B V V Prasad andM K Estes ldquoStructural requirements for the assembly of Nor-walk virus-like particlesrdquo Journal of Virology vol 76 no 8 pp4044ndash4055 2002

[13] B V PrasadM E Hardy T Dokland J Bella M G Rossmannand M K Estes ldquoX-ray crystallographic structure of the Nor-walk virus capsidrdquo Science vol 286 no 5438 pp 287ndash290 1999

[14] A Bertolotti-Ciarlet S E Crawford A M Hutson and M KEstes ldquoThe 31015840 end of norwalk virus mRNA contains determi-nants that regulate the expression and stability of the viral capsidprotein vp1 a novel function for the vp2 proteinrdquo Journal ofVirology vol 77 no 21 pp 11603ndash11615 2003

[15] T N Hoa Tran E Trainor T Nakagomi N A Cunliffe and ONakagomi ldquoMolecular epidemiology of noroviruses associatedwith acute sporadic gastroenteritis in children global distri-bution of genogroups genotypes and GII4 variantsrdquo Journal ofClinical Virology vol 56 no 3 pp 185ndash193 2013

[16] K Green ldquoCaliciviridae the norovirusesrdquo in Field VirologyD M Knipe and P M Howley Eds pp 582ndash608 LippincottWilliams ampWilkins Philadelphia Pa USA 6th edition 2013

[17] S Fukuda Y Sasaki S Takao andM Seno ldquoRecombinant nor-ovirus implicated in gastroenteritis outbreaks in HiroshimaPrefecture Japanrdquo Journal of Medical Virology vol 80 no 5 pp921ndash928 2008

[18] F C Tseng J S Leon J NMacCormack J-MMaillard and CL Moe ldquoMolecular epidemiology of norovirus gastroenteritisoutbreaks inNorth Carolina United States 1995ndash2000rdquo Journalof Medical Virology vol 79 no 1 pp 84ndash91 2007

[19] T-H Han S-C Kim S-T Kim C-H Chung and J-Y ChungldquoDetection of norovirus genogroup IV klassevirus and peppermild mottle virus in sewage samples in South Koreardquo Archivesof Virology vol 159 no 3 pp 457ndash463 2014

[20] Y Yan H-H Wang L Gao et al ldquoA one-step multiplex real-time RT-PCR assay for rapid and simultaneous detection ofhuman norovirus genogroup I II and IVrdquo Journal of VirologicalMethods vol 189 no 2 pp 277ndash282 2013

[21] J J Siebenga H Vennema B Renckens et al ldquoEpochal evolu-tion of GGII4 norovirus capsid proteins from 1995 to 2006rdquoJournal of Virology vol 81 no 18 pp 9932ndash9941 2007

[22] A Polkowska M Ronnqvist and O Lepisto ldquoOutbreak of gas-troenteritis caused by norovirus GII4 Sydney variant after awedding reception at a resortactivity centre Finland August2012rdquo Epidemiology and Infection vol 142 no 9 pp 1877ndash18832012

[23] S M Ahmed B A Lopman and K Levy ldquoA systematic reviewand meta-analysis of the global seasonality of norovirusrdquo PLoSONE vol 8 no 10 Article ID e75922 2013

[24] J A Marshall and L D Bruggink ldquoThe dynamics of norovirusoutbreak epidemics recent insightsrdquo International Journal ofEnvironmental Research and Public Health vol 8 no 4 pp 1141ndash1149 2011

[25] H S Kim J HyunH-S Kim J-S KimW Song andKM LeeldquoEmergence of GII4 Sydney norovirus in South Korea duringthe winter of 2012-2013rdquo Journal of Microbiology and Biotech-nology vol 23 no 11 pp 1641ndash1643 2013

[26] S H Kim D S Cheon J H Kim et al ldquoOutbreaks of gastro-enteritis that occurred during school excursions in Korea wereassociatedwith several waterborne strains of norovirusrdquo Journalof Clinical Microbiology vol 43 no 9 pp 4836ndash4839 2005

[27] DHafligerM Gilgen J Luthy and PHubner ldquoSeminested RT-PCR systems for small round structured viruses and detection

of enteric viruses in seafoodrdquo International Journal of FoodMicrobiology vol 37 no 1 pp 27ndash36 1997

[28] EWollants andM van Ranst ldquoDetection of false positives witha commonly used Norovirus RT-PCR primer setrdquo Journal ofClinical Virology vol 56 no 1 pp 84ndash85 2013

[29] L Kittigul A Panjangampatthana K Pombubpa et al ldquoDetec-tion and genetic characterization of norovirus in environmentalwater samples in Thailandrdquo The Southeast Asian Journal ofTropical Medicine and Public Health vol 43 no 2 pp 323ndash3322012

[30] H S Lee and J H Lim SPSS 200 Manual Jiphyunjae SeoulRepublic of Korea 2013

[31] A J Hall B A Lopman D C Payne et al ldquoNorovirus diseasein the united statesrdquo Emerging Infectious Diseases vol 19 no 8pp 1198ndash1205 2013


[33] H F RabenauM Sturmer S Buxbaum AWalczokW Preiserand H W Doerr ldquoLaboratory diagnosis of norovirus whichmethod is the bestrdquo Intervirology vol 46 no 4 pp 232ndash2382003

[34] J Vinje ldquoAdvances in laboratory methods for detection andtyping of norovirusrdquo Journal of Clinical Microbiology 2014

[35] E de Bruin E Duizer H Vennema and M P G KoopmansldquoDiagnosis of Norovirus outbreaks by commercial ELISA orRT-PCRrdquo Journal of Virological Methods vol 137 no 2 pp 259ndash264 2006

[36] M Swellam M S Mahmoud and A A-F Ali ldquoDiagnosis ofhepatitis C virus infection by enzyme-linked immunosorbentassay and reverse transcriptase-nested polymerase chain reac-tion a comparative evaluationrdquo IUBMB Life vol 63 no 6 pp430ndash434 2011

[37] E Guevremont J Brassard A Houde C Simard and Y-LTrottier ldquoDevelopment of an extraction and concentration pro-cedure and comparison of RT-PCR primer systems for thedetection of hepatitis A virus and norovirus GII in greenonionsrdquo Journal of VirologicalMethods vol 134 no 1-2 pp 130ndash135 2006

[38] I L A Boxman J J H C Tilburg N A J M Te Loeke et alldquoDetection of noroviruses in shellfish in the Netherlandsrdquo Inter-national Journal of Food Microbiology vol 108 no 3 pp 391ndash396 2006

[39] HK Joung SHHan S-J Park et al ldquoNationwide surveillancefor pathogenic microorganisms in groundwater near carcassburials constructed in South Korea in 2010rdquo InternationalJournal of Environmental Research and Public Health vol 10 no12 pp 7126ndash7143 2013

[40] S-G Lee W-H Jheong C-I Suh et al ldquoNationwide ground-water surveillance of noroviruses in SouthKorea 2008rdquoAppliedand Environmental Microbiology vol 77 no 4 pp 1466ndash14742011

[41] J-G Fu J Ai X Qi J Zhang F-Y Tang and Y-F Zhu ldquoEmer-gence of two novel norovirus genotype II4 variants associatedwith viral gastroenteritis in Chinardquo Journal of Medical Virologyvol 86 no 7 pp 1226ndash1234 2014

[42] H Mai M Jin X Guo et al ldquoClinical and epidemiologic cha-racteristics of norovirus GII4 sydney during winter 2012-13 inbeijing china following its global emergencerdquo PLoS ONE vol8 no 8 Article ID e71483 2013


[43] A Thongprachum W Chan-it P Khamrin et al ldquoMolecularepidemiology of norovirus associated with gastroenteritis andemergence of norovirus GII4 variant 2012 in Japanese pediatricpatientsrdquo Infection Genetics and Evolution vol 23 pp 65ndash732014

[44] J Fonager S Barzinci and T K Fischer ldquoEmergence of a newrecombinant Sydney 2012 norovirus variant in Denmark 26December 2012 to 22 March 2013rdquo Eurosurveillance vol 18 no25 2013

[45] J-S EdenMM TanakaM F BoniW D Rawlinson and P AWhite ldquoRecombination within the pandemic norovirus GII4lineagerdquo Journal of Virology vol 87 no 11 pp 6270ndash6282 2013

[46] V Martella M C Medici S de Grazia et al ldquoEvidence forrecombination between pandemic GII4 norovirus strains NewOrleans 2009 and Sydney 2012rdquo Journal of ClinicalMicrobiologyvol 51 no 11 pp 3855ndash3857 2013

[47] M C W Chan T F Leung A K Kwok N Lee and P K SChan ldquoCharacteristics of patients infected with norovirus GII4Sydney 2012 Hong Kong Chinardquo Emerging Infectious Diseasesvol 20 no 4 pp 558ndash661 2014

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table 1 Description of NV GI sequences for genetic analysis




EU085529 GI1 P774Delsjo2004GothenburgSweden KF039731 GI1 CH4XO5332009USA

FJ515294 GI2 Leuven2003BEL KF039732 GI1 CHA5A0102009USA

JN176918 GI2 Roosendaal0292006NL KF039733 GI1 CHA9A004 201104262011USA

JN183159 GI9 S482008Lilla KF039734 GI1 CHA6A0142009USA

JN183161 GI7 S242008Lilla Edet KF039735 GI1 CHA3A0072008USA

JN603244 GI3 S292008Lilla EdetSweden KF039736 GI1 CHA7A0112010USA

JN603245 GI4 S502008Lilla EdetSweden KF039737 GI1 CHA6A003 200911042009USA

JQ388274 GI6 KingstonACT160D2010AU KF306212 GI2 Jingzhou2013401CHN

JQ743331 GI4 2000 KF429761 GI1 8MoIIIL1972USA

JQ743332 GI2 1999 KF429765 GI1 8W1968USA

JQ911594 GI 103602010VNM KF429770 GI1 8McIII1973USA

JX023285 GI1 8FIIa1968USA KF429773 GI1 8CKIIIc1974USA

KC998959 GI6 TCH-099USA2003 KF429774 GI1 8UIIIf1973USA

KF039725 GI1 CHA7A0092010USA KF429783 GI1 8K1979USA

KF039726 GI1 CHA6A003 200910312009USA KF429789 GI1 8MC1978USA

KF039727 GI1 CHA2A0142008USA KF586507 GI9 CAIQ12110628

KF039728 GI1 CHA2A0142008USA L07418KF039729 GI1 CHA6A0072010USA M87661KF039730 GI1 CHA9A004 201104192011USA

strain is difficult with existing RT-PCR primer sets (GII-F1R1F2 SRII-123) because of the continuous variation ofthe strain [26 27] In addition GI-F1R1 primer set does notalways have sufficient specificity to detect NV because false-positive detection commonly occurs [28]

Therefore the aim of this studywas to develop primer setsfor efficiently detectingNVGI andGII including detection ofnewly emerged strains that could not previously be identifiedwith conventional primer sets Once new primer sets weredeveloped we evaluated their efficiency using an RT-PCRassay to test clinical and environmental specimens

2 Materials and Methods

21 Collection of Clinical and Environmental Samples Twosample types clinical and environmental specimens wereused for detection of NV GI and GII Eighty-six unknownsamples were used for detection of NV GI They included22 clinical samples from Gyeonggi Institute of Health Envi-ronment (GIHE) that were originally obtained during 2012and 2013 and 24 clinical and 40 environmental samples fromWaterborne Virus Bank (WAVA) originally obtained from2006 to 2013 To identify NVGII we used 134 unknown sam-ples that included 35 clinical samples from GIHE originallycollected during 2012 and 2013 and 32 clinical and 67 envi-ronmental samples fromWAVA collected from 2006 to 2013

All stool specimens were collected from patients whosuffered from diarrhea caused by acute gastroenteritis Envi-ronmental specimenswere collected fromgroundwater in theRepublic of Korea during June and October 2013 All sampleswere stored at minus80∘C until use

22 Ethical Clearance All clinical samples were obtainedduring themedical treatment of patients with acute gastroen-teritis All patients providedwritten informed consent whichhas been kept on file at the GIHE and WAVA Human rightswere not abused nor were ethical issues encountered duringthe study All experimental work and collection of sampleswere supervised and approved by the Institutional ReviewBoard (IRB) of SongeuiMedical CampusTheCatholic Univ-ersity of Korea (approval number MC14SISI0039)

23 Primer Design In order to design new primer sets37 sequences of NV GI (Table 1) and 52 sequences of NVGII (Table 2) were obtained from NCBI and imported intoEditSeq and MegAlign in DNASTAR software (DNASTARUSA)

We designed 3 candidates for the NV GI primer set and 5candidates for theNVGII primer set in the conserved regionsof ORF 1 and ORF 2 Among them NKI and NKII (Table 3)which have outstanding efficiency were selected Inner pri-mers were designed to detect NV fromwater samples because















(a) GI

NKI primer set





(b) GII

NKII primer set








Capsid 51+



Capsid 55+



Capsid 50minus





RdRp 55+






RdRp 50minus






3 Results








3998400

5998400

RdRp


GI-F1R1 (5342ndash5671)

ORF1 ORF2 ORF3

(a)

3998400

5998400

RdRp



ORF1 ORF2 ORF3

(b)















4 Discussion







5 Conclusions





Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















(a) GI

NKI primer set





(b) GII

NKII primer set








Capsid 51+



Capsid 55+



Capsid 50minus





RdRp 55+






RdRp 50minus






3 Results








3998400

5998400

RdRp


GI-F1R1 (5342ndash5671)

ORF1 ORF2 ORF3

(a)

3998400

5998400

RdRp



ORF1 ORF2 ORF3

(b)















4 Discussion







5 Conclusions





Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



(a) GI

NKI primer set





(b) GII

NKII primer set








Capsid 51+



Capsid 55+



Capsid 50minus





RdRp 55+






RdRp 50minus






3 Results








3998400

5998400

RdRp


GI-F1R1 (5342ndash5671)

ORF1 ORF2 ORF3

(a)

3998400

5998400

RdRp



ORF1 ORF2 ORF3

(b)















4 Discussion







5 Conclusions





Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Capsid 51+



Capsid 55+



Capsid 50minus





RdRp 55+






RdRp 50minus






3 Results








3998400

5998400

RdRp


GI-F1R1 (5342ndash5671)

ORF1 ORF2 ORF3

(a)

3998400

5998400

RdRp



ORF1 ORF2 ORF3

(b)















4 Discussion







5 Conclusions





Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


3998400

5998400

RdRp


GI-F1R1 (5342ndash5671)

ORF1 ORF2 ORF3

(a)

3998400

5998400

RdRp



ORF1 ORF2 ORF3

(b)















4 Discussion







5 Conclusions





Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






5 Conclusions





Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Development of Enhanced Primer Sets for