Colorimetric Detection of Norovirus in Oysters Samples through

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Colorimetric Detection of Norovirus in Oysters Samples through DNAzyme as Signaling Probe Bhagwan Sahebrao Batule, Seong U Kim, Hyoyoung Mun, Changsun Choi, Won-Bo Shim, and Min-Gon Kim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05289 • Publication Date (Web): 30 Jan 2018 Downloaded from http://pubs.acs.org on January 31, 2018

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Journal of Agricultural and Food Chemistry

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Colorimetric Detection of Norovirus in Oysters Samples through DNAzyme as Signaling

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Probe

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Bhagwan S Batule,a Seong U Kim,b Hyoyoung Mun,a Changsun Choi,c Won-Bo Shim,d

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Min-Gon Kima,b*

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a

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Technology, 261 Chemdan-gwagiro, Gwangju 500-712, Republic of Korea

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b

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Chemdan-gwagiro, Gwangju 500-712, Republic of Korea

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Department of Chemistry, School of Physics and Chemistry, Gwangju Institute of Science and

Advanced Photonics Research Institute, Gwangju Institute of Science and Technology, 261

Department of Agricultural chemistry and Food Science and Technology, Gyeongsang National

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University, 900 Gajwa-dong Jinju Gyeongnam 660-701, Republic of Korea

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d

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Ang University, Ansung-Si, Gyounggi, 17546, South Korea

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*Corresponding author: E-mail address: [email protected] (M.G. Kim), TEL: +82-62-715-3330,

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FAX: +82-62-715-3419

Department of Food and Nutrition, College of Biotechnology and Natural Resources, Chung-

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1 ACS Paragon Plus Environment


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Abstract

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Worldwide, norovirus (NV) is one of the most associated cause of acute gastroenteritis, which

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leads nearly 50,000 child deaths every year in developing countries. Therefore, there is great

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demand to develop rapid, low-cost, and accurate detection assay for the foodborne norovirus

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infection in order to reduce mortality caused by norovirus. Considering importance of norovirus,

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we have demonstrated a highly sensitive and specific colorimetric detection method for analysis

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of human norovirus genogroups I and II (HuNoV GI and II) in oyster samples. This is the first

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report to employing colorimetric HRPzyme-integrated PCR for direct norovirus detection from

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the real shellfish samples. We found that the HRPzyme-integrated PCR method is more sensitive

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than gel-electrophoresis approach and could detect HuNoV GI and II genome upto one copy per

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milliliter. The specificity of the proposed method was successfully demonstrated for HuNoV GI

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and II. Further, we performed testing HuNoVs in the spiked oyster samples and the HRPzyme-

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integrated PCR method proved to be an ultrasensitive and selective method for detecting

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HuNoVs in the real samples. By integrating the proposed method with the portable PCR machine,

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it would be more reliable to improve food safety by detecting HuNoVs in the different types of

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shellfish such as oyster and mussel at the production field.

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Keywords: norovirus; PCR; G-quadruplex; shellfish; colorimetric sensor

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Introduction

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Noroviruses are now recognized as the most common foodborne pathogen causing diarrhea,

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acute gastroenteritis, and food poisoning in people of all ages.1-3 Noroviruses can survive in

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diverse environmental conditions and it is often transmitted via ingestion of contaminated water

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or food, direct contact, or inhalation of aerosols.4-6 The genogroups (GI and II) of noroviruses


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contain the most human-specific strains.7 In 2006, 31 school cafeterias, 2,400 students were

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infected by norovirus food poisoning in Korea.8 Recent studies revealed that noroviruses are

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present in seafood such as shellfish and oyster.9,10 The contamination of oyster with noroviruses

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is a serious public health issue.11 Therefore, early detection of clinical infection noroviruses is

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important as it can facilitate more rapid implementation of rigorous controls, which can result in

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reduced health care costs and improved public health.

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Several research groups reported detection methods for norovirus such as Enzyme-linked

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immunosorbent assay (ELISA),12 antibody-based magnetic separation,13 dipstick immunoassay,14

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drop microfluidic system,15 isothermal amplification,16 and DNA aptasensor.17 Real-time reverse

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transcription-polymerase chain reaction (RT-PCR)-based methods are employed as a gold

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standard method to detect HuNoV GI and II RNA from sea-food samples.18,19 Still,

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aforementioned methods need costly chemicals, advanced equipment, and expert assistants to

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perform the analysis.20-22 Considering the limited applicability of RT-PCR on-site detection of

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norovirus, there is great demand to develop rapid and reliable methods for on-site detection of

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HuNoVs.

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Recently, different colorimetric detection platforms have been developed based on horseradish

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peroxidase-mimicking DNAzyme (HRPzyme) as a signaling probe for detection different

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analytes such as viruses and bacteria.23,24 The HRPzyme is composed of a guanine (G)-rich

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sequence, which fold into G-quadruplex structure in the presence of hemin and catalyze

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peroxidation with 2,2’-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and H2O2.25,26

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The HRPzyme showed several characteristics such as robust, inexpensive, and naked-eye

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detection.27 Recently, HRPzyme integrated primers have been employed as a colorimetric probe

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in the PCR amplification for sensitive and selective detection of pathogenic bacteria.28-30 3 ACS Paragon Plus Environment


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Therefore, HRPzyme-integrated PCR would be ideal for the detection of norovirus. In this study,

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we have developed a novel approach for rapid and precise detection of food-borne norovirus

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based on HRPzyme integrated PCR. To the best our knowledge, this is the first report using

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HRPzyme integrated primers based colorimetric PCR for HuNoV GI and GII in the spiked and

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real sample of oyster.

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Materials and methods:

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Materials and reagents

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ABTS and Hemin were purchased from Sigma-Aldrich (Yongin, South Korea). Tris-buffered

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ethylenediaminetetraacetic acid buffer was obtained from LPS Solution (Daejeon, South Korea).

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Thermus thermophilus (Tth) DNA polymerase was purchased from Epicentre Technologies

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(Madison, WI, USA). The 100-bp DNA Ladder (Dye Plus) was purchased from Takara Bio

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(Seoul, South Korea). All primers used in this study were purchased from Genotech (Daejeon,

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South Korea). Agarose powder was purchased from Roche (Seoul, South Korea). All reagents

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and solvents used in this study were of analytical grade and utilized without further purification.

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The primer sets employed for norovirus detection are presented in Table 1.

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Sensitivity of norovirus detection

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We utilized most important HuNoV GI and GII genotypes as detection analytes.19 Based on

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highly homogenous region of GI and GII was utilized to select HRPzyme-integrated PCR

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primers using primer3 software. PCR reactions were performed at optimized conditions in a total

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volume of 50 µL containing 5 µL of norovirus suspensions at different concentrations (0 to 103

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copies per reaction), 2.5 µL of 10× PCR buffer, 6 µL of 25 mM MgCl2, 8 µL of 2.5 mM dNTP

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mix, 5 µL of 10× PCR enhancer, 1 µL of 25 mM MnSO4, 1 µL of 20 µM forward primer, 1 µL 4 ACS Paragon Plus Environment

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of 20 µM reverse primer, and 0.5 µL of Tth DNA polymerase. PCR was performed under the

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following conditions: denaturation at 95 °C for 5 min and at 60 °C for 20 min, followed by 35

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cycles of denaturation at 94 °C for 10 s, annealing at 60 °C for 25 s, and primer extension at

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72 °C for 10 s. The final extension was performed at 72 °C for 1 min. Control PCR was

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performed in the absence of target norovirus copies. First, PCR-based amplification of norovirus

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with specific primers (F and R) was confirmed by electrophoresis in a 1.5% agarose gel. Then, at

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the end of the amplification, 10 µL of the PCR product, 5.5 µL of 300 µM hemin, 100 µL of 5.5

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mM ABTS, 0.7 µL of 35% H2O2, and 1 mL of citrate buffer (pH 4) were mixed and the

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absorbance was measured at 410 nm after 10 min at room temperature with a microplate reader

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(Infinite M2000pro, TECAN group, Ltd., Switzerland) or images were captured with a digital

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camera (Samsung, seoul, south korea).

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Detection of norovirus in spiked oyster samples

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First, we collected aliquots of pancreatic tissues obtained from control oysters samples, which

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were tested as a negative for HuNoVs (GI and GII). Then, 200 µL dilutions of norovirus

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suspension were utilized for artificial contamination of oyster samples. The samples were

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vortexed for 1 min before preparing of different diluted virus concentrations. Three replicates

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were prepared from dilution and used for further experiment such as feasibility and sensitivity

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studies. The detection procedure was used as same as mentioned above.

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Instrumentation

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The absorbance of the ABTS oxidation product was determined using an Infinite 1000 reader

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(Tecan, Mannedorf, Switzerland) at 410 nm. PCR amplification reactions were performed on a

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C1000 thermal cycler (Bio-Rad, Hercules, CA, USA). Images were captured with an NX 200



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digital camera (Samsung, Seoul, South Korea). The relative intensity (a.u.) value was obtained

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by subtracting the average intensity of the negative samples from the average intensity of the

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positive samples.

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Result and discussion:

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Principle and design of colorimetric detection of HuNoVs

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In this work, one-step colorimetric HRPzyme-integrated PCR method was successfully utilized

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for the detection of HuNoVs. The primers (forward and reverse) were modified with four regions,

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such as protector, HRPzyme, spacer and the complementary sequence to norovirus RNA. In our

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previous report,28 we have optimized size of protector and spacer sequence. Based on HRPzyme-

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integrated primers, we have developed a sensitive and selective colorimetric detection method

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for HuNoVs (Figure 1). The protector and spacer sequences employed for blocking the folding

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of HRPzyme sequence in the double strand case and avoiding nonspecific binding. But, the

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protector sequence did not showed any negative effect on catalytic activity of folded-HRPzyme

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sequence. Therefore, blocked HRPzyme could not produces color signal in the presence of

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ABTS and H2O2, and vice versa. Based on previous reports,22 we designed primers for HuNoV

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GI and GII, which are mentioned in the Table 1. We have amplified HuNoV genes in one-step

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PCR. First, we lysed virus particles by boiling samples containing HuNoVs. Then, we employed

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thermostable Tth DNA polymerase from Thermus thermophilus, which have reverse

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transcriptase as well as polymerase activity. Further, cDNA synthesis and PCR amplification

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simultaneously was carried-out in one-step. In the presence of norovirus, double stranded

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products were generated through HRPzyme-integrated PCR, then blocked HRPzyme could not


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fold into a quadruplex structure in the presence of hemin. In the absence of norovirus,

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unamplified primers folded into G-quadruplex with hemin molecule. Further, the catalytic

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hemin-G-quadruplex complex catalyzes the oxidation of ABTS in the presence of H2O2 and

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produces colored product.29-31

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Optimization of primer concentration and protector sequence length

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Based on the colorimetric signal, we have tested effect of primer concentration and protector

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poly adenine (A) nucleotides (ntds) length. First, we employed different concentration of primer

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(10 - 800 nM) and found that 400 nM exhibited highest relative intensity (Figure S1a). Then, we

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tested different length of poly-A (0 – 20 ntds) as protector nucleotides sequence at 5’ end of

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primers (forward and reverse primers) and found that 10 ntds showed maximum relative

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intensity (Figure S1b). Based on previous report,28 poly-A nucleotides showed least effect on G-

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quadruplex folding and catalytic activity. We employed these optimized conditions for the

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further experiments.

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Feasibility study of the proposed colorimetric method

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With optimized conditions, we have demonstrated our proposed strategy by successfully

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detecting HuNoV GI and II in (Figure 2). We have utilized optimized conditions from our

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previous report.28 For this purpose, we designed HuNoV GI and II specific primers (forward and

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reverse), containing target specific, spacer, HRPzyme, and protector sequence at 3′ end. We

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performed PCR amplification of HuNoV GI and II with modified primers presented in the Table

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1. First, we confirmed PCR amplification of HuNoV GI and II gene product through 1.5%

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agarose gel electrophoresis (Figure 2a). In Figure 2a, the bands in lanes 2 and 4 correspond to

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samples HuNoV GI and II, respectively. Further, the colorimetric signal was generated by



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addition hemin, H2O2, and ABTS, which can be confirmed by naked eyes (Figure 2b) and

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corresponding quantitative data are presented in the Figure 2c. Notably, PCR reaction without

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target did not showed any specific bands but solution turned to blue-green color due to catalytic

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activity of unamplified primers. There was a significant difference between with and without

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norovirus sample for sensitive detection.

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Sensitivity study of the proposed strategy for HuNoV GI and II detection

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We have successfully quantified HuNoV GI and II based on our proposed strategy by detecting

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different concentrations. First, we prepared different concentrations of HuNoV GI and II with

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range 0 to 104 copies mL-1 diluted in the PBS buffer. Further, The PCR amplification was

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performed with different concentrations of norovirus particles without RNA extraction. The PCR

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amplification of HuNoV GI and II confirmed by agarose gel electrophoresis. In figure 3a and 4a,

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the band intensities were directly proportional to concentrations of respective HuNoV GI and II.

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The colorimetric signals were inversely proportional to the concentration of respective HuNoV

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GI and II (Figure 3b and 4b). The figure 3c and 4c represent the quantitative data of color

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intensities. The color intensities of PCR products depict negative linear relationship with the

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concentration of HuNoV GI and II (Figure 3c and 4c). The standardization curve of color

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intensities versus different concentrations of the HuNoV GI and II showed excellent linearity in

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the range of concentrations from 1.0 to 1.0 × 104 copies mL-1 (Figure 3c). Our colorimetric

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HRPzyme-integrated PCR detection platform was exhibited 10-fold (HuNoV GI) and 100 fold

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(HuNoV GII) better result than the agarose gel-electrophoresis based assay. Further, the

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proposed strategy of HuNoVs detection was compared with some previously reported methods

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(Table S1). Compare with previous methods of HuNoVs detection, our proposed method showed


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several advantages such as one-tube two step, cost-effective, genomic RNA extraction free,

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naked eyes detection.

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Further, we successfully verified the selectivity of the proposed strategy for the detection of

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HuNoV GI and II with different pathogenic bacteria (such as Escherichia coli O157:H7, Listeria

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monocytogenes, Vibrio parahaemolyticus, Salmonella enterica Typhimurium, and Bacillus

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cereus), which are commonly contaminated at low levels in oyster samples.32 Based on the

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aforementioned optimized conditions, we selectively detected HuNoV GI and II with other

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pathogenic bacteria with concentration 104 cfu mL-1 and found that there were obtained

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negligible colorimetric intensity with non-target pathogenic bacteria, and vice versa (Figure S2).

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The results showed the excellent specificity of the proposed strategy towards HuNoV GI and II

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detection. In addition, there are many ions (such as Fe, Cu, Co, and Pt), which can affect the

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color change of peroxidase substrate (such as ABTS, 3, 3′, 5, 5′-tetramethylbenzidine (TMB),

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and amplex ultra red (AUR)) by H2O2.33-36 Importantly, the nanomolar concentration of metal

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ions in the biological sample does not produce any colorimetric signal in the presence of ABTS.

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Therefore, in our experiments, we did not found any interference of metal ions such as Fe, Cu,

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Co, and Pt on the final colorimetric signal of oxidized ABTS.

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Detection of HuNoV GI and II in the spiked oyster sample

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Recently, many cases were reported due to consumption of norovirus infected raw or under-

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cooked oysters or shellfish. Therefore, the detection of norovirus from fresh oysters or shellfish

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is more important to stop spreading of norovirus infection. In this work, we have validated the

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applicability of the proposed colorimetric detection method for norovirus in the spiked oyster

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samples, which were purchased from the local market. We have spiked known concentration of 9 ACS Paragon Plus Environment


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HuNoV GI and II (0 to 103 copies mL-1) in oyster samples. The accuracy and precision of

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proposed HRPzyme-integrated PCR were estimated by using recovery analysis from the spiked

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oyster samples, which were obtained from the local market. Satisfactory recoveries of G I and II

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in the range 92 to 105% were obtained after samples analysis with three replicates (Table 2).

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Further, we detected norovirus from the HuNoV-infected oyster real samples. The HuNoV-

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infected oyster obtained from obtained from korean sea. We directly tested a real sample with

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our proposed method and successfully detected HuNoV GI and II (Table 3). Further, we

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investigated reliability our proposed method for real oyster samples from the local market along

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with the conventional real-time PCR method. The table 3 represents that our method showed

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good agreement with conventional method. Moreover, this proposed method has several

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advantages such as label-free, low-cost, naked-eyes, and point-of-care diagnosis.

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Detection of norovirus is clinical important due to many norovirus infected cases showed acute

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gastroenteritis and also reported morbidity worldwide. Therefore, we developed a simple,

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sensitive and selective reverse-signaling based colorimetric HRPzyme-integrated PCR. In this

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strategy, we have designed integrated primer containing four regions such as target specific,

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spacer, HRPzyme, and protector sequence. We have successfully detected norovirus upto single

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with suitable detection range through colorimetric signal generated by HRPzyme-hemin complex

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mediated oxidation of ABTS with H2O2. In addition, we have confirmed diagnostic capability by

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detection norovirus in spiked as well as real oyster sample. By integrating this proposed strategy

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with the portable PCR machine could be applied in the detection of norovirus in resource-limited

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area.

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Conflicts of interest

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There are no conflicts to declare. 10 ACS Paragon Plus Environment

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Acknowledgments

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1. This research was supported by a grant from the World Institute of Kimchi funded by the

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Ministry of Science, ICT and Future Planning (KE1701-5), Republic of Korea.

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2. This research was supported by the GIST (Gwangju Institute of Science and Technololgy),

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Korea, under the Practical Research and Development support program supervised by the

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GTI (GIST Technology Institute), Republic of Korea.

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Supporting information available:

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[The figure S1 represents optimization of colorimetric detection of noroviruses. (a) Primer

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concentration; (b) Protector sequence length. The figure S2 represents specificity of proposed

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strategy towards HuNoVs GI and GII.

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monocytogenes; 4, Vibrio parahaemolyticus; 5, Salmonella Typhimurium; 6, Bacillus cereus.

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The concentration of HuNoVs GI, HuNoVs GII, and all bacteria were 103, 103, and104 cfu mL−1,

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respectively. Error bars are represent the standard deviations from three representative

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experiments (n = 3). The table S1 indicates the comparison of proposed strategy with other

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reported methods for the detection of HuNoV. All supporting information are presented and

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available as a free of charge via internet at http://pubs.ac.org.]

1, HuNoVs; 2, E. coli O157:H7; 3, Listeria

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one-step rapid visual detection of bisphenol A in water samples by label-free aptasensor.

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Biosens. Bioelectron. 2013, 39, 26-30.

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Kim, S. U.; Batule, B. S.; Mun, H.; Shim, W. B.; Kim, M. G., Ultrasensitive colorimetric

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detection of salmonella enterica Typhimurium on lettuce leaves by HRPzyme-integrated

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polymerase chain reaction. Food Control 2017, 84, 522-528.



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DePaola, A.; Jones, J. L.; Woods, J.; Burkhardt, W.; Calci, K. R.; Krantz, J. A.; Bowers, J.

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2007 United States market survey. Appl. Environ. Microbiol. 2010, 76, 2754-2768.

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protein-inorganic nanoflowers. Int. J. Nanomedicine 2015, 10, 137.

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Batule, B. S.; Park, K. S.; Kim, M. I.; Park, H. G., Ultrafast sonochemical synthesis of

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Qin, Y.; Zhang, L.; Ye, G.; Zhao, S., Homogeneous label-free colorimetric strategy for

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convenient bleomycin detection based on bleomycin enhanced Fe(II)–H2O2–ABTS

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reaction. Anal. Methods 2014, 6, 7973-7977.

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Shu, J.; Qiu, Z.; Wei, Q.; Zhuang, J.; Tang, D., Cobalt-porphyrin-platinum-functionalized

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reduced graphene oxide hybrid nanostructures: A novel peroxidase mimetic system for

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improved electrochemical immunoassay. Sci. Rep. 2015, 5, 15113.

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36.

Valekar, A. H.; Batule, B. S.; Kim, M. I.; Cho, K.-H.; Hong, D.Y.; Lee, U.H.; Chang, J.S.; Park, H. G.; Hwang, Y. K., Novel amine-functionalized iron trimesates with enhanced


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peroxidase-like activity and their applications for the fluorescent assay of choline and

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acetylcholine. Biosens. Bioelectron. 2018, 100, 161-168.

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Figure caption

359

Figure 1. Schematic Illustration of the HRPzyme-based colorimetric PCR method for norovirus

360

detection from the oyster sample

361

Figure 2. Feasibility study for detection of HuNoV GI and II. a) The electrophoresis data of 1.

362

With HuNoV GI, 2. Without HuNoV GI, 3. With HuNoV GII and 4. Without HuNoV GII; b)

363

The colorimetric images of 1. With HuNoV GI, 2. Without HuNoV GI, 3. With HuNoV GII and

364

4. Without HuNoV GII; c) The quantitative data of (b). Each data represents the mean values ±

365

standard deviation for replicates (n=3).

366

Figure 3. Sensitivity of HuNoV G I. (a) Gel electrophoresis data of different concentrations of

367

GI. (b) naked-eye detection of G I and (c) calibration curve toward GI. Each data represents the

368

mean values ± standard deviation for replicates (n=3).

369

Figure 4. Sensitivity of HuNoV G II. (a) Gel electrophoresis data of different concentrations of

370

GII. (b) naked-eye detection of GII and (c) calibration curve toward GII. Each data represents the

371

mean values ± standard deviation for replicates (n=3).

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374

375

376

377

378

Table 1 Primers information of HRPzyme-integrated PCR utilized for detection of norovirus

379

(HuNoV) genogroup I (GI) and genogroup II (GII). HuNoV

Target

detection

region

HuNoV

RdRp

Primers

primer sequence (5′ → 3′)a

GI-Fb

AAAAAAAAAAGGGTAGGGCGGGTTGGGTAAAAA

GI

CGYTGGATGCGITTYCATGA GI-Rb

AAAAAAAAAAGGGTAGGGCGGGTTGGGTAAAAA CTTAGACGCCATCATCATTYAC

HuNoV

BPO

GII-Fb

GII

AAAAAAAAAAGGGTAGGGCGGGTTGGGTAAAAA AICCIATGTTYAGITGGATGAG

GII-Rb

AAAAAAAAAAGGGTAGGGCGGGTTGGGTAAAAA TCGACGCCATCTTCATTCACA

380

a

Mixed bases in degenerate primers as follows: Y = C or T; I = inosine;

381

b

Primers as described by Kageyama et al. (2003).22

382


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383

384

385

386

387

388

Table 2. Recovery study of HuNoV GI and II in spiked oyster sample (ո = 3). ND: Not detected.

HuNoV

Sample

Added (copies)

Found (copies)

Recovery (%)

1

0

ND

2

1 x 101

(0.9258 ± 0.020 ) x 101

92.58

HuNoV

3

1 x 102

(0.9645 ± 0.035) x 102

96.45

GI

4

1 x 103

(1.0008 ± 0.053) x 103

100.08

1

0

ND

HuNoV

2

1 x 101

(1.0051 ± 0.058) x 101

100.51

GII

3

1 x 102

(0.9811 ± 0.042) x 102

98.11

4

1 x 103

(1.0844 ± 0.049) x 103

108.44

No.

389

390

391

Table 3. Detection of Real oyster samples obtained from Korean sea



Samples

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Real time PCR (copies)

Our proposed method (copies)

GI

GI

S1

GII 8.45

S2

94.26

S3

1041.14

GII 11.27

100.01 142.22

995.50

146.33

392

393

394

395

Figure 1

396

397

398

399


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401

402

403

404

405

406

407

408

Figure 2

409

410

411 412



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413

414

415

416

417

418

419

420

Figure 3

421


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422 423

424

425

Figure 4



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427 428

429

430

TOC Graphic

431

(category: Analytical methods; Food safety and toxicology)


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434

435


Colorimetric Detection of Norovirus in Oysters Samples through

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