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RNA extraction method for the PCR detection of hepatitis A virus in shellfish
International Journal of Food Microbiology 142 (2010) 198–201
Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
RNA extraction method for the PCR detection of hepatitis A virus in shellfish Valentina Terio ⁎, Angela Di Pinto, Pietro Di Pinto, Vito Martella, Giuseppina Tantillo Università degli Studi di Bari, Prov. le per Casamassima, km 3, 70010 Valenzano, Bari, Italy
a r t i c l e
i n f o
Article history: Received 18 February 2010 Received in revised form 23 June 2010 Accepted 27 June 2010 Keywords: Shellfish HAV RNA extraction RT-PCR hn-PCR
a b s t r a c t Viruses are the leading cause of foodborne illness associated with the consumption of raw or slightly-cooked contaminated shellfish. This study evaluated the E.Z.N.A. Mollusc RNA extraction and purification kit for the detection of HAV in shellfish. The E.Z.N.A. method, based on the cationic detergent, cetyltrimethyl ammonium bromide, in conjunction with a selective RNA binding silica matrix, efficiently isolated viral RNA with a detection limit of 1 TCID50/ml by hemi-nested PCR. This method proved to be faster and less expensive than PEG precipitation-based procedures. It is also technically undemanding, requiring no extensive processing steps or excess manipulation, minimizing RNA degradation and ensuring the absence of PCR inhibitors. The E.Z.N.A. method applied to HAV screening of shellfish samples from the Apulian region, revealed a high level of contamination. These results confirm that conventional faecal indicators are unreliable for demonstrating the presence or absence of viruses. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Enteric viruses are a significant public health problem in many countries, due to the persistent circulation of such viruses in the environment and the possible contamination of water and food. These viruses are the leading cause of foodborne illness in the USA (Mead et al., 1999) and viral agents have been responsible for a significant number of disease outbreaks in Europe (de Wit et al., 2003). In south-eastern Italy, HAV infection is still endemic, with annual incidence rates of up to 30 cases/100,000, and recurrent outbreaks occur associated with raw seafood consumption. In the Apulia region, a major epidemic was reported in 1996 and 1997, with more than 5000 cases associated with shellfish consumption. Person–person transmission was an important risk factor in the second phase of the outbreak, from 1996 to 1997 (Germinario et al., 2000; Lo Palco et al., 2005). Epidemiological studies indicate that filter-feeding shellfish are important vectors for the transmission of enteric viral diseases, due to their tendency to concentrate waterborne pathogens (Richards, 1985). In southern Italy, mussels are a major risk factor for HAV because they are often eaten raw or slightlycooked (Mele et al., 1989, 1994). In particular, slightly-cooked mussels are heated for only a few minutes until the valves open. Experimental studies indicate that HAV can remain infectious within mussel tissues unless thoroughly cooked (Croci et al., 2005).
⁎ Corresponding author. Dipartimento di Sanità Pubblica e Zootecnia — Sezione Controllo e Sicurezza degli Alimenti, Facoltà di Medicina Veterinaria, Università degli Studi di Bari; Prov. le per Casamassima, km 3, 70010 Valenzano, Bari, Italy. Tel.: + 390805443970; fax: + 390805443855. E-mail address: [email protected] (V. Terio). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.06.026
The European guidelines in EU Regulation 2073/2005 base shellfish safety exclusively on analysis for specific bacteriological parameters, Salmonella and Escherichia coli, which do not correlate with the presence of viruses, according to Goyal et al. (1979). Previous observations have shown that conventional depuration systems are unable to eliminate viruses completely, as these may persist in shellfish for several days (De Medici et al., 2001; Sobsey et al., 1988). The European Scientific Committee on Veterinary Measures relating to Public Health (SCVMPH) concluded that the conventional faecal indicators are unreliable for demonstrating the presence or absence of enteric viruses and that the reliance on faecal bacterial indicator removal for determining shellfish purification times is an unsafe practice (EC Regulation 2073/2005). HAV risk assessment of shellfish requires the development and the availability of specific and sensitive analytical systems (Lees, 2000) to evaluate the potential hazard for public health. In the last few years, extraction and molecular detection methods have been developed for detecting viruses (Le Guyader et al., 1996; Cromeans et al., 1997; Kingsley and Richards, 2001; Mullendore et al., 2001; Romalde et al., 2002). Although PCR-based assays are extremely sensitive, they can be limited by the presence of natural PCR inhibitors in shellfish samples. Thus, successful molecular analyses are critically dependent on RNA extraction and purification methods. Considering that the establishment of standard methods for detecting viruses in mussels is a priority for EC Regulation 2073/2005, the aim of this study was to evaluate the E.Z.N.A. Mollusc RNA kit (Omega Bio-tek, USA) both on experimentally- and on naturallycontaminated shellfish. This viral RNA extraction and purification method is based on the properties of the cationic detergent, cetyltrimethyl ammonium bromide (CTAB), in conjunction with the selective RNA binding of a specific matrix.
V. Terio et al. / International Journal of Food Microbiology 142 (2010) 198–201
2. Materials and methods
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(hn-PCR) (Le Guyader et al., 1994). The primers were synthesized by PRIMM (Milan, Italy).
2.1. Reference strain and shellfish sample seeding 2.5. RT-PCR The HAV reference strain 8° free K-4, a cell culture-adapted and cytopathic strain propagated in Fetal Rhesus Kidney-4 derived (FRhK4) cells, with a titer of 106 TCID50/ml, was used as a positive control. 10-fold dilutions of viral suspension, carried out with Dulbecco's Modified Eagle medium (DMEM), were performed and used to experimentally contaminate the shellfish samples. In particular, Mytilus galloprovincialis samples that previously tested negative for HAV were scrubbed with a brush and washed with sterile distilled water. They were then aseptically shucked with a sterilized knife and the hepatopancreas from 10 of these shellfish (30 mg) was homogenized using a sterile pestle, contaminated with 0.1 ml of each serial 10-fold dilution of the HAV reference strain (from 10− 1 to 10− 8) and crushed up with liquid nitrogen and sterile pestle in a Motor Cordless homogeniser (Kontes Glass Company, Vineland, New Jersey). 2.2. Sampling The E.Z.N.A. method Mollusc RNA kit (Omega Bio-tek, USA) was used to test 118 M. galloprovincialis samples, collected between July 2008 and April 2009 from Apulian shellfish depuration plants after standard purification practices at 13 °C for 8 h. The shellfish samples were stored at 4 °C and transferred to the laboratory for processing within 4 h. The samples were analyzed for E. coli and Salmonella, performed according to EC Regulation 2073/2005. Next, the hepatopancreas from 10 M. galloprovincialis (30 mg) were analyzed for HAV using the molecular method. Mussel samples testing negative for HAV were subjected to spiking test with 1%, 10%, 20%, 40%, 60%, and 80% of HAV reference RNA (106 TCID50/ml) and retested to investigate the possible presence of RT-PCR inhibitors. 2.3. Viral RNA extraction The shellfish hepatopancreas homogenates from experimentally contaminated samples, together with samples collected from Apulian shellfish depuration plants, were subjected to viral RNA extraction performed using the E.Z.N.A. Mollusc RNA kit (Omega Bio-tek, USA). Then 1 additional hepatopancreas sample was not spiked and used as a negative control. Samples were added to 350 μl Buffer MRL/2mercaptoethanol (Merck, Germany) and were vortexed vigorously. The lysate was further added to 350 μl chloroform:isoamyl alcohol (24:1) and centrifuged at 12,000 g for 2 min at room temperature. After adding one volume of isopropanol to the aqueous phase, the RNA precipitation was obtained by centrifugation at 13,000 g for 2 min. The pellet was resuspended with 100 μl of sterile DEPC-treated water preheated to 65 °C and then incubated at 65 °C for 2 min. After adjusting the binding conditions by adding 350 μl Buffer RB/2mercaptoethanol (Merck, Germany) and 250 μl absolute ethanol, the mixture was centrifuged at 12,000 g for 15 s at room temperature, and then washed to improve the RNA purity by centrifugation steps at 12,000 g for 15 s, adding first 500 μl Wash Buffer I and then 500 μl Wash Buffer II. 2.4. Oligonucleotide primers The study used the upstream primer 5′-GTT TTG CTC CTC TTT ATC ATG CTA TG-3′ (2167–2193) and as the downstream primer 5′-GGA AAT GTC TCA GGT ACT TTC TTG-3′ (2389–2414), as previously described (Le Guyader et al., 1994), which produced a 248 bp long fragment located within the conserved region VP3–VP1 of the HAV genome. An internal primer, HAV3: 5′-TCC TCA ATT GTT GTG ATA GC3′, producing a 210 bp long fragment, was used for hemi-nested PCR
Reverse transcription polymerase chain reaction (RT-PCR) was carried out in a single step using SuperScriptTM One Step RT-PCR with Platinum Taq (INVITROGEN Life Technologies, Milan, Italy). All components for RT-PCR were assembled in a 25 μl reaction with 12.5 μl 2× Reaction Mix, a buffer containing 0.4 mM of each dNTP and 2.4 mM MgSO4, 2 μl of RT/Platinum Taq Mix, 0.5 μM of each specific gene primer and 3 μl of template RNA. Positive control with purified HAV RNA was added to verify the PCR reaction. Also negative control (no RNA) was included to check reagent cross contamination. Subsequently, cDNA synthesis was carried out by incubation for 15 min at 50 °C. PCR was performed with an initial denaturation step at 95 °C for 5 min and 35 cycles of 95 °C for 15 s, 53 °C for 30 s, and 70 °C for 30 s. RT-PCR was programmed in a Mastercycler (Eppendorf, Milan, Italy). 2.6. Hemi-nested PCR The hn-PCR was carried out on experimentally contaminated samples and 118 samples collected from Apulian shellfish depuration plants. The reaction was performed in a final volume of 25 μl using 12.5 μl of HotStarTaq Master Mix (QIAGEN, Hilden, Germany), which provides 2.5 units per reaction of DNA Polymerase, 0.2 mM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dTTP), 1× PCR buffer (with 1.5 mM-MgCl2). PCR was performed with 3 μl of RT-PCR product, diluted 1:100. The mixture was processed with an initial denaturation step of 95 °C for 15 min, followed by 35 cycles of denaturation at 94 °C for 30 sc, annealing at 50 °C for 30 s, extension at 72 °C for 30 s, with a final extension at 72 °C for 5 min. Finally, 210 bp products were subjected to sequence analysis. Sequence analysis of PCR products was carried out by ABI PRISM 3100 (Applied Biosystems, Rome, Italy). 2.7. Detection of amplified products The RT-PCR and hn-PCR products were analyzed by electrophoresis on 1.5% (w/v) agarose NA gel (Pharmacia, Uppsala, Sweden) in 1× TBE buffer containing 0.89 M Tris, 0.89 M boric acid, 0.02 M EDTA, pH 8.0 (USB, Cleveland, Ohio, USA) and visualized through ethidium bromide staining under UV-transilluminator. A Gene RulerTM 100 bp DNA Ladder Plus (MBI Fermentas, Vilnius, Lithuania) was used as the molecular weight marker. Image acquisition was performed using UVIPro Gel-Documentation (UVItec, Cambridge, UK). 3. Results 3.1. Viral RNA extraction The E.Z.N.A. Mollusc RNA kit (Omega Bio-tek, USA), based on the reversible binding properties of a silica matrix, is time saving and allows the simultaneous processing of multiple samples. In a single working day, up to 30 samples may be processed. The E.Z.N.A. method reduces the quantity of starting material and requires common equipment with low analysis costs (4.00 € per sample). 3.2. RT-PCR RT-PCR, carried out on experimentally contaminated shellfish samples, showed viral RNA suitable for RT-PCR. Sensitivity evaluation gave a detection limit of 10 TCID50/ml after one round of amplification (RT-PCR). This RT-PCR produced amplicons of the expected sizes (248 bp). No non-specific products were observed.
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3.3. Hemi-nested PCR (hn-PCR) A second amplification by hn-PCR produced a detection limit 1 logarithmic unit higher than RT-PCR (1 TCID50/ml), producing a specific 210 bp product (Fig. 1). The results shown in Fig. 1 are representative of five independent experiments with no aspecific reactions observed. 3.4. Sampling The 118 shellfish samples showed bacteriological parameters (E. coli and Salmonella) compliant with EC Regulation 2073/2005. The presence of the HAV genome was detected in 17/118 samples using RT-PCR (Fig. 2) and in 71/118 samples with hn-PCR (Fig. 3). The specificity of amplicons was verified by sequence analysis and corresponded to the published sequence of the HAV strain. The spiking test highlighted different RT-PCR inhibitor concentrations in only 7/47 negative samples. In particular, 4/7 negative samples showed a signal at 20% HAV reference strain while 3/7 highlighted a signal at 40% HAV reference strain. 4. Discussion and conclusions Because of the lack of standardized methods for detecting viruses in shellfish, many “in-house” and commercial methods are available (Croci et al., 1999; Sair et al., 2002; Di Pinto et al., 2003; Ribao et al., 2004; Suñén et al., 2004). Most analytical methods for viral detection are based on sampling of whole mussel and polyethylene glycol (PEG) precipitation, a suitable method for concentrating the low virus levels present in food (Di Pinto et al., 2003; Ribao et al., 2004; Suñén et al., 2004; Kingsley and Richards, 2003). Secondly, the extraction and purification methods currently used include phenol/chloroform, TRIzol and commercial kits, which are based on the use of resins and organic solvents (Di Pinto et al., 2003; Ribao et al., 2004; Suñén et al., 2004; Kingsley and Richards, 2003). The E.Z.N.A. Mollusc RNA kit (Omega Bio-tek, USA) is a rapid and highly efficient RNA extraction and purification method, providing an opportunity for molecular detection of viral agents and for the prevention of foodborne viral diseases (Desenclos et al., 1991; Le Guyader et al., 1994; Croci et al., 1999; Sair et al., 2002; Lee et al., 1999). Briefly, the method includes shellfish hepatopancreas homogenization, viral RNA extraction and purification based on the
Fig. 1. Electrophoretic profile of hn-PCR products from experimentally contaminated shellfish samples. Lane 1: 100 bp DNA Ladder; Lane 2: positive sample (210 bp); Lane 3: 103 TCID50/ml; Lane 4: 102 TCID50/ml; Lane 5: 10 TCID50/ml; Lane 6: 1 TCID50/ml; Lane 7: negative sample; Lane 8: negative sample (no RNA).
Fig. 2. Electrophoretic profile of RT-PCR products from naturally-contaminated shellfish samples. Lane 1: negative control (no RNA); Lane 2: positive control; Lanes 3, 4, 5, 6, and 7: RT-PCR sample; Lane 8: 100 bp DNA Ladder.
properties of CTAB, in conjunction with the selective RNA binding of a specific silica matrix, followed by RT-nested-PCR. The E.Z.N.A. method afforded suitable viral RNA recovery and good removal of inhibitory substances, resulting in efficient RT-PCR detection of viral RNA. Indeed, among several viral RNA extraction methods, such as guanidinium isothiocyanate, TRIzol, microspin column, CTAB, GTCsilica, and Chelex, the use of a silica matrix was found to be the most effective at purifying RNA from complex sample matrices, according to Sair et al. (2002). Furthermore, this method avoids the use of reagents able to inhibit RT-PCR. The E.Z.N.A. method is not technically demanding, because it does not require extensive processing steps and avoids excess manipulation, minimizing RNA degradation and the presence of natural RT-PCR inhibitors. Compared with the other
Fig. 3. Electrophoretic profile of hn-PCR products from naturally-contaminated shellfish samples. Lane 1: negative control (no RNA); Lane 2: hn-PCR negative control; Lane 3: hn-PCR positive control; Lanes 4 and 5: hn-PCR samples; Lane 6: 100 bp DNA Ladder.
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procedures, this method proved faster and less expensive. Indeed, the E.Z.N.A. method is less time-consuming than previous approaches based on PEG precipitation (Kingsley and Richards, 2003; Croci et al., 2000; Goswami et al., 2002; Di Pinto et al., 2004). Also, the procedure is not particularly costly, being less expensive than the system previously described by Di Pinto et al. (2003, 2004) based on the use of the FastTrack 2.0 mRNA Isolation Kit (Invitrogen, Carlsbad, USA) making it a more useful application for routine monitoring. The application of the E.Z.N.A. method on the shellfish hepatopancreas, a major source of virus particles (Kingsley and Richards, 2003; Schwab et al., 1998), appears interesting in virological analysis. However, accurate comparisons between these methods are not possible because of the differences related to parameters such as virus titer units of reference viral suspension. The E.Z.N.A. method, applied to HAV screening of shellfish samples from the Apulia region, revealed a high level of contamination according to Chironna et al. (2002). The discrepancies observed between the RT-PCR and the hn-PCR results were probably due to low levels of viral particles in mussels or to the presence of natural inhibitors. The study confirms that conventional faecal indicators are unreliable for demonstrating the presence or absence of viruses, according to the Scientific Committee on Veterinary Measures relating to Public Health (SCVMPH). Moreover, this study proves further evidence that conventional depuration systems cannot guarantee shellfish virological safety and public health, due to long-term virus persistence in shellfish (De Medici et al., 2001; Le Guyader et al., 2006). The development of reliable methods for viral hazards is necessary to establish specific criteria for pathogenic viruses according to EC Regulation 2073/2005. The PCR-based methods are efficient, sensitive, specific and time-saving approaches applicable for the detection of viruses in shellfish to acquire data on HAV prevalence. Also, the high consumption of raw or slightly-cooked mussels in southern Italy requires a systematic and continuous gathering, analysis, interpretation and distribution of health data, including epidemiological studies on viral hepatitis. The E.Z.N.A. method may be used for a single mass screening to implement specific control strategies necessary for the shellfish trade within the EU and for exports outside of the EU, because it enables up to 30 samples per day to be carried out. The method is only qualitative and provides no information on viral infectivity. A further aim is to perform the integration of cell culture with PCR (ICC-PCR) to acquire specific information on the infectivity of a virus (Croci et al, 2003; Griffin et al., 2003), although HAV in cell culture systems, however, is much slower and nonlytic and does not produce a detectable cytopathic effect in infected cells (Koch and Koch, 1985). References Chironna, M., Germinario, C., De Medici, D., Fiore, A., Di Pasquale, S., Quarto, M., Barbuti, S., 2002. Detection of hepatitis A virus in mussels from different sources marketed in Puglia region (south Italy). International Journal of Food Microbiology 75, 11–18. Croci, L., De Medici, D., Morace, G., Fiore, A., Scalfaro, C., Benedice, F., Toti, L., 1999. Detection of hepatitis A virus in shellfish by nested reverse transcription-PCR. International Journal of Food Microbiology 48, 67–71. Croci, L., De Medici, D., Scalfaro, C., Fiore, A., Dovizia, M., Donia, D., Casentino, A.M., Moretti, P., Costantini, G., 2000. Determination of enterovirus, hepatitis A virus; bacteriophages and E. coli in Adriatic Sea mussels. Journal of Applied Microbiology 88, 293–298. Croci, L., De Medici, D., Ciccozzi, M., Di Pasquale, S., Suffredini, E., Toti, L., 2003. Contamination of mussels by hepatitis A virus: a public health problem in southern Italy. Food Control 4, 559–563. Croci, L., De Medici, D., Di Pasquale, S., Toti, L., 2005. Resistance of hepatitis A virus in mussels subjected to different domestic cookings. International Journal of Food Microbiology 105, 139–144. Cromeans, T.L., Nainan, O.V., Margolis, H.S., 1997. Detection of hepatitis A virus RNA in oyster meat. Applied and Environmental Microbiology 63, 2460–2463. De Medici, D., Ciccozzi, M., Fiore, A., Di Pasquale, S., Parlato, A., Ricci-Bitti, P., Croci, L., 2001. Closed-circuit system for the depuration of mussels experimentally contaminated with hepatitis A virus. Journal of Food Protection 64, 877–880.
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Contents lists available at ScienceDirect
International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
RNA extraction method for the PCR detection of hepatitis A virus in shellfish Valentina Terio ⁎, Angela Di Pinto, Pietro Di Pinto, Vito Martella, Giuseppina Tantillo Università degli Studi di Bari, Prov. le per Casamassima, km 3, 70010 Valenzano, Bari, Italy
a r t i c l e
i n f o
Article history: Received 18 February 2010 Received in revised form 23 June 2010 Accepted 27 June 2010 Keywords: Shellfish HAV RNA extraction RT-PCR hn-PCR
a b s t r a c t Viruses are the leading cause of foodborne illness associated with the consumption of raw or slightly-cooked contaminated shellfish. This study evaluated the E.Z.N.A. Mollusc RNA extraction and purification kit for the detection of HAV in shellfish. The E.Z.N.A. method, based on the cationic detergent, cetyltrimethyl ammonium bromide, in conjunction with a selective RNA binding silica matrix, efficiently isolated viral RNA with a detection limit of 1 TCID50/ml by hemi-nested PCR. This method proved to be faster and less expensive than PEG precipitation-based procedures. It is also technically undemanding, requiring no extensive processing steps or excess manipulation, minimizing RNA degradation and ensuring the absence of PCR inhibitors. The E.Z.N.A. method applied to HAV screening of shellfish samples from the Apulian region, revealed a high level of contamination. These results confirm that conventional faecal indicators are unreliable for demonstrating the presence or absence of viruses. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Enteric viruses are a significant public health problem in many countries, due to the persistent circulation of such viruses in the environment and the possible contamination of water and food. These viruses are the leading cause of foodborne illness in the USA (Mead et al., 1999) and viral agents have been responsible for a significant number of disease outbreaks in Europe (de Wit et al., 2003). In south-eastern Italy, HAV infection is still endemic, with annual incidence rates of up to 30 cases/100,000, and recurrent outbreaks occur associated with raw seafood consumption. In the Apulia region, a major epidemic was reported in 1996 and 1997, with more than 5000 cases associated with shellfish consumption. Person–person transmission was an important risk factor in the second phase of the outbreak, from 1996 to 1997 (Germinario et al., 2000; Lo Palco et al., 2005). Epidemiological studies indicate that filter-feeding shellfish are important vectors for the transmission of enteric viral diseases, due to their tendency to concentrate waterborne pathogens (Richards, 1985). In southern Italy, mussels are a major risk factor for HAV because they are often eaten raw or slightlycooked (Mele et al., 1989, 1994). In particular, slightly-cooked mussels are heated for only a few minutes until the valves open. Experimental studies indicate that HAV can remain infectious within mussel tissues unless thoroughly cooked (Croci et al., 2005).
⁎ Corresponding author. Dipartimento di Sanità Pubblica e Zootecnia — Sezione Controllo e Sicurezza degli Alimenti, Facoltà di Medicina Veterinaria, Università degli Studi di Bari; Prov. le per Casamassima, km 3, 70010 Valenzano, Bari, Italy. Tel.: + 390805443970; fax: + 390805443855. E-mail address: [email protected] (V. Terio). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.06.026
The European guidelines in EU Regulation 2073/2005 base shellfish safety exclusively on analysis for specific bacteriological parameters, Salmonella and Escherichia coli, which do not correlate with the presence of viruses, according to Goyal et al. (1979). Previous observations have shown that conventional depuration systems are unable to eliminate viruses completely, as these may persist in shellfish for several days (De Medici et al., 2001; Sobsey et al., 1988). The European Scientific Committee on Veterinary Measures relating to Public Health (SCVMPH) concluded that the conventional faecal indicators are unreliable for demonstrating the presence or absence of enteric viruses and that the reliance on faecal bacterial indicator removal for determining shellfish purification times is an unsafe practice (EC Regulation 2073/2005). HAV risk assessment of shellfish requires the development and the availability of specific and sensitive analytical systems (Lees, 2000) to evaluate the potential hazard for public health. In the last few years, extraction and molecular detection methods have been developed for detecting viruses (Le Guyader et al., 1996; Cromeans et al., 1997; Kingsley and Richards, 2001; Mullendore et al., 2001; Romalde et al., 2002). Although PCR-based assays are extremely sensitive, they can be limited by the presence of natural PCR inhibitors in shellfish samples. Thus, successful molecular analyses are critically dependent on RNA extraction and purification methods. Considering that the establishment of standard methods for detecting viruses in mussels is a priority for EC Regulation 2073/2005, the aim of this study was to evaluate the E.Z.N.A. Mollusc RNA kit (Omega Bio-tek, USA) both on experimentally- and on naturallycontaminated shellfish. This viral RNA extraction and purification method is based on the properties of the cationic detergent, cetyltrimethyl ammonium bromide (CTAB), in conjunction with the selective RNA binding of a specific matrix.
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2. Materials and methods
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(hn-PCR) (Le Guyader et al., 1994). The primers were synthesized by PRIMM (Milan, Italy).
2.1. Reference strain and shellfish sample seeding 2.5. RT-PCR The HAV reference strain 8° free K-4, a cell culture-adapted and cytopathic strain propagated in Fetal Rhesus Kidney-4 derived (FRhK4) cells, with a titer of 106 TCID50/ml, was used as a positive control. 10-fold dilutions of viral suspension, carried out with Dulbecco's Modified Eagle medium (DMEM), were performed and used to experimentally contaminate the shellfish samples. In particular, Mytilus galloprovincialis samples that previously tested negative for HAV were scrubbed with a brush and washed with sterile distilled water. They were then aseptically shucked with a sterilized knife and the hepatopancreas from 10 of these shellfish (30 mg) was homogenized using a sterile pestle, contaminated with 0.1 ml of each serial 10-fold dilution of the HAV reference strain (from 10− 1 to 10− 8) and crushed up with liquid nitrogen and sterile pestle in a Motor Cordless homogeniser (Kontes Glass Company, Vineland, New Jersey). 2.2. Sampling The E.Z.N.A. method Mollusc RNA kit (Omega Bio-tek, USA) was used to test 118 M. galloprovincialis samples, collected between July 2008 and April 2009 from Apulian shellfish depuration plants after standard purification practices at 13 °C for 8 h. The shellfish samples were stored at 4 °C and transferred to the laboratory for processing within 4 h. The samples were analyzed for E. coli and Salmonella, performed according to EC Regulation 2073/2005. Next, the hepatopancreas from 10 M. galloprovincialis (30 mg) were analyzed for HAV using the molecular method. Mussel samples testing negative for HAV were subjected to spiking test with 1%, 10%, 20%, 40%, 60%, and 80% of HAV reference RNA (106 TCID50/ml) and retested to investigate the possible presence of RT-PCR inhibitors. 2.3. Viral RNA extraction The shellfish hepatopancreas homogenates from experimentally contaminated samples, together with samples collected from Apulian shellfish depuration plants, were subjected to viral RNA extraction performed using the E.Z.N.A. Mollusc RNA kit (Omega Bio-tek, USA). Then 1 additional hepatopancreas sample was not spiked and used as a negative control. Samples were added to 350 μl Buffer MRL/2mercaptoethanol (Merck, Germany) and were vortexed vigorously. The lysate was further added to 350 μl chloroform:isoamyl alcohol (24:1) and centrifuged at 12,000 g for 2 min at room temperature. After adding one volume of isopropanol to the aqueous phase, the RNA precipitation was obtained by centrifugation at 13,000 g for 2 min. The pellet was resuspended with 100 μl of sterile DEPC-treated water preheated to 65 °C and then incubated at 65 °C for 2 min. After adjusting the binding conditions by adding 350 μl Buffer RB/2mercaptoethanol (Merck, Germany) and 250 μl absolute ethanol, the mixture was centrifuged at 12,000 g for 15 s at room temperature, and then washed to improve the RNA purity by centrifugation steps at 12,000 g for 15 s, adding first 500 μl Wash Buffer I and then 500 μl Wash Buffer II. 2.4. Oligonucleotide primers The study used the upstream primer 5′-GTT TTG CTC CTC TTT ATC ATG CTA TG-3′ (2167–2193) and as the downstream primer 5′-GGA AAT GTC TCA GGT ACT TTC TTG-3′ (2389–2414), as previously described (Le Guyader et al., 1994), which produced a 248 bp long fragment located within the conserved region VP3–VP1 of the HAV genome. An internal primer, HAV3: 5′-TCC TCA ATT GTT GTG ATA GC3′, producing a 210 bp long fragment, was used for hemi-nested PCR
Reverse transcription polymerase chain reaction (RT-PCR) was carried out in a single step using SuperScriptTM One Step RT-PCR with Platinum Taq (INVITROGEN Life Technologies, Milan, Italy). All components for RT-PCR were assembled in a 25 μl reaction with 12.5 μl 2× Reaction Mix, a buffer containing 0.4 mM of each dNTP and 2.4 mM MgSO4, 2 μl of RT/Platinum Taq Mix, 0.5 μM of each specific gene primer and 3 μl of template RNA. Positive control with purified HAV RNA was added to verify the PCR reaction. Also negative control (no RNA) was included to check reagent cross contamination. Subsequently, cDNA synthesis was carried out by incubation for 15 min at 50 °C. PCR was performed with an initial denaturation step at 95 °C for 5 min and 35 cycles of 95 °C for 15 s, 53 °C for 30 s, and 70 °C for 30 s. RT-PCR was programmed in a Mastercycler (Eppendorf, Milan, Italy). 2.6. Hemi-nested PCR The hn-PCR was carried out on experimentally contaminated samples and 118 samples collected from Apulian shellfish depuration plants. The reaction was performed in a final volume of 25 μl using 12.5 μl of HotStarTaq Master Mix (QIAGEN, Hilden, Germany), which provides 2.5 units per reaction of DNA Polymerase, 0.2 mM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, and dTTP), 1× PCR buffer (with 1.5 mM-MgCl2). PCR was performed with 3 μl of RT-PCR product, diluted 1:100. The mixture was processed with an initial denaturation step of 95 °C for 15 min, followed by 35 cycles of denaturation at 94 °C for 30 sc, annealing at 50 °C for 30 s, extension at 72 °C for 30 s, with a final extension at 72 °C for 5 min. Finally, 210 bp products were subjected to sequence analysis. Sequence analysis of PCR products was carried out by ABI PRISM 3100 (Applied Biosystems, Rome, Italy). 2.7. Detection of amplified products The RT-PCR and hn-PCR products were analyzed by electrophoresis on 1.5% (w/v) agarose NA gel (Pharmacia, Uppsala, Sweden) in 1× TBE buffer containing 0.89 M Tris, 0.89 M boric acid, 0.02 M EDTA, pH 8.0 (USB, Cleveland, Ohio, USA) and visualized through ethidium bromide staining under UV-transilluminator. A Gene RulerTM 100 bp DNA Ladder Plus (MBI Fermentas, Vilnius, Lithuania) was used as the molecular weight marker. Image acquisition was performed using UVIPro Gel-Documentation (UVItec, Cambridge, UK). 3. Results 3.1. Viral RNA extraction The E.Z.N.A. Mollusc RNA kit (Omega Bio-tek, USA), based on the reversible binding properties of a silica matrix, is time saving and allows the simultaneous processing of multiple samples. In a single working day, up to 30 samples may be processed. The E.Z.N.A. method reduces the quantity of starting material and requires common equipment with low analysis costs (4.00 € per sample). 3.2. RT-PCR RT-PCR, carried out on experimentally contaminated shellfish samples, showed viral RNA suitable for RT-PCR. Sensitivity evaluation gave a detection limit of 10 TCID50/ml after one round of amplification (RT-PCR). This RT-PCR produced amplicons of the expected sizes (248 bp). No non-specific products were observed.
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3.3. Hemi-nested PCR (hn-PCR) A second amplification by hn-PCR produced a detection limit 1 logarithmic unit higher than RT-PCR (1 TCID50/ml), producing a specific 210 bp product (Fig. 1). The results shown in Fig. 1 are representative of five independent experiments with no aspecific reactions observed. 3.4. Sampling The 118 shellfish samples showed bacteriological parameters (E. coli and Salmonella) compliant with EC Regulation 2073/2005. The presence of the HAV genome was detected in 17/118 samples using RT-PCR (Fig. 2) and in 71/118 samples with hn-PCR (Fig. 3). The specificity of amplicons was verified by sequence analysis and corresponded to the published sequence of the HAV strain. The spiking test highlighted different RT-PCR inhibitor concentrations in only 7/47 negative samples. In particular, 4/7 negative samples showed a signal at 20% HAV reference strain while 3/7 highlighted a signal at 40% HAV reference strain. 4. Discussion and conclusions Because of the lack of standardized methods for detecting viruses in shellfish, many “in-house” and commercial methods are available (Croci et al., 1999; Sair et al., 2002; Di Pinto et al., 2003; Ribao et al., 2004; Suñén et al., 2004). Most analytical methods for viral detection are based on sampling of whole mussel and polyethylene glycol (PEG) precipitation, a suitable method for concentrating the low virus levels present in food (Di Pinto et al., 2003; Ribao et al., 2004; Suñén et al., 2004; Kingsley and Richards, 2003). Secondly, the extraction and purification methods currently used include phenol/chloroform, TRIzol and commercial kits, which are based on the use of resins and organic solvents (Di Pinto et al., 2003; Ribao et al., 2004; Suñén et al., 2004; Kingsley and Richards, 2003). The E.Z.N.A. Mollusc RNA kit (Omega Bio-tek, USA) is a rapid and highly efficient RNA extraction and purification method, providing an opportunity for molecular detection of viral agents and for the prevention of foodborne viral diseases (Desenclos et al., 1991; Le Guyader et al., 1994; Croci et al., 1999; Sair et al., 2002; Lee et al., 1999). Briefly, the method includes shellfish hepatopancreas homogenization, viral RNA extraction and purification based on the
Fig. 1. Electrophoretic profile of hn-PCR products from experimentally contaminated shellfish samples. Lane 1: 100 bp DNA Ladder; Lane 2: positive sample (210 bp); Lane 3: 103 TCID50/ml; Lane 4: 102 TCID50/ml; Lane 5: 10 TCID50/ml; Lane 6: 1 TCID50/ml; Lane 7: negative sample; Lane 8: negative sample (no RNA).
Fig. 2. Electrophoretic profile of RT-PCR products from naturally-contaminated shellfish samples. Lane 1: negative control (no RNA); Lane 2: positive control; Lanes 3, 4, 5, 6, and 7: RT-PCR sample; Lane 8: 100 bp DNA Ladder.
properties of CTAB, in conjunction with the selective RNA binding of a specific silica matrix, followed by RT-nested-PCR. The E.Z.N.A. method afforded suitable viral RNA recovery and good removal of inhibitory substances, resulting in efficient RT-PCR detection of viral RNA. Indeed, among several viral RNA extraction methods, such as guanidinium isothiocyanate, TRIzol, microspin column, CTAB, GTCsilica, and Chelex, the use of a silica matrix was found to be the most effective at purifying RNA from complex sample matrices, according to Sair et al. (2002). Furthermore, this method avoids the use of reagents able to inhibit RT-PCR. The E.Z.N.A. method is not technically demanding, because it does not require extensive processing steps and avoids excess manipulation, minimizing RNA degradation and the presence of natural RT-PCR inhibitors. Compared with the other
Fig. 3. Electrophoretic profile of hn-PCR products from naturally-contaminated shellfish samples. Lane 1: negative control (no RNA); Lane 2: hn-PCR negative control; Lane 3: hn-PCR positive control; Lanes 4 and 5: hn-PCR samples; Lane 6: 100 bp DNA Ladder.
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procedures, this method proved faster and less expensive. Indeed, the E.Z.N.A. method is less time-consuming than previous approaches based on PEG precipitation (Kingsley and Richards, 2003; Croci et al., 2000; Goswami et al., 2002; Di Pinto et al., 2004). Also, the procedure is not particularly costly, being less expensive than the system previously described by Di Pinto et al. (2003, 2004) based on the use of the FastTrack 2.0 mRNA Isolation Kit (Invitrogen, Carlsbad, USA) making it a more useful application for routine monitoring. The application of the E.Z.N.A. method on the shellfish hepatopancreas, a major source of virus particles (Kingsley and Richards, 2003; Schwab et al., 1998), appears interesting in virological analysis. However, accurate comparisons between these methods are not possible because of the differences related to parameters such as virus titer units of reference viral suspension. The E.Z.N.A. method, applied to HAV screening of shellfish samples from the Apulia region, revealed a high level of contamination according to Chironna et al. (2002). The discrepancies observed between the RT-PCR and the hn-PCR results were probably due to low levels of viral particles in mussels or to the presence of natural inhibitors. The study confirms that conventional faecal indicators are unreliable for demonstrating the presence or absence of viruses, according to the Scientific Committee on Veterinary Measures relating to Public Health (SCVMPH). Moreover, this study proves further evidence that conventional depuration systems cannot guarantee shellfish virological safety and public health, due to long-term virus persistence in shellfish (De Medici et al., 2001; Le Guyader et al., 2006). The development of reliable methods for viral hazards is necessary to establish specific criteria for pathogenic viruses according to EC Regulation 2073/2005. The PCR-based methods are efficient, sensitive, specific and time-saving approaches applicable for the detection of viruses in shellfish to acquire data on HAV prevalence. Also, the high consumption of raw or slightly-cooked mussels in southern Italy requires a systematic and continuous gathering, analysis, interpretation and distribution of health data, including epidemiological studies on viral hepatitis. The E.Z.N.A. method may be used for a single mass screening to implement specific control strategies necessary for the shellfish trade within the EU and for exports outside of the EU, because it enables up to 30 samples per day to be carried out. The method is only qualitative and provides no information on viral infectivity. A further aim is to perform the integration of cell culture with PCR (ICC-PCR) to acquire specific information on the infectivity of a virus (Croci et al, 2003; Griffin et al., 2003), although HAV in cell culture systems, however, is much slower and nonlytic and does not produce a detectable cytopathic effect in infected cells (Koch and Koch, 1985). References Chironna, M., Germinario, C., De Medici, D., Fiore, A., Di Pasquale, S., Quarto, M., Barbuti, S., 2002. Detection of hepatitis A virus in mussels from different sources marketed in Puglia region (south Italy). International Journal of Food Microbiology 75, 11–18. Croci, L., De Medici, D., Morace, G., Fiore, A., Scalfaro, C., Benedice, F., Toti, L., 1999. Detection of hepatitis A virus in shellfish by nested reverse transcription-PCR. International Journal of Food Microbiology 48, 67–71. Croci, L., De Medici, D., Scalfaro, C., Fiore, A., Dovizia, M., Donia, D., Casentino, A.M., Moretti, P., Costantini, G., 2000. Determination of enterovirus, hepatitis A virus; bacteriophages and E. coli in Adriatic Sea mussels. Journal of Applied Microbiology 88, 293–298. Croci, L., De Medici, D., Ciccozzi, M., Di Pasquale, S., Suffredini, E., Toti, L., 2003. Contamination of mussels by hepatitis A virus: a public health problem in southern Italy. Food Control 4, 559–563. Croci, L., De Medici, D., Di Pasquale, S., Toti, L., 2005. Resistance of hepatitis A virus in mussels subjected to different domestic cookings. International Journal of Food Microbiology 105, 139–144. Cromeans, T.L., Nainan, O.V., Margolis, H.S., 1997. Detection of hepatitis A virus RNA in oyster meat. Applied and Environmental Microbiology 63, 2460–2463. De Medici, D., Ciccozzi, M., Fiore, A., Di Pasquale, S., Parlato, A., Ricci-Bitti, P., Croci, L., 2001. Closed-circuit system for the depuration of mussels experimentally contaminated with hepatitis A virus. Journal of Food Protection 64, 877–880.
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