Applied and Environmental Microbiology, October 2004, p . 6329-6332, Vol . 70, No . 10

Reverse Transcription-Booster PCR for Detection of Noroviruses in Shellfish

Dario De Medici,^1* Luciana Croci,¹ Elisabetta Suffredini,¹ and Laura Toti¹

Laboratorio Alimenti, Istituto Superiore di Sanità, Rome, Italy¹

Received 10 November 2003/ Accepted 12 June 2004

ABSTRACT

The methods commonly used for norovirus (NV) detection are based on reverse transcription-PCR (RT-PCR) followed by confirmation of the amplified sequence . To increase sensitivity, an RT-booster PCR was developed . The proposed method showed an increase in sensitivity at least 2 log units for all the NV strains tested compared with the standard RT-PCR method . Higher sensitivity was confirmed in tests on experimentally and naturally contaminated shellfish .

Noroviruses (NVs) represent the most important cause of viral gastroenteritis outbreaks worldwide (11, 14, 15) . Food- and waterborne transmission of NVs is common (5), and different epidemiological studies have implicated NVs in outbreaks linked to shellfish consumption (2) . In many outbreaks of shellfish-transmitted diseases, however, the causative agent has not been identified due to the lack of a sensitive method of analysis of shellfish samples (12) . In fact, as the levels of enteric viruses in mussels are normally low, high-sensitivity detection methods are usually needed .

Different strategies of reverse transcription-PCR (RT-PCR) to enable the molecular detection of the largest number of NV strains possible have been proposed (3) . An effective approach to improve the chances of successful amplification consists of associating primers selected in a mostly conserved region (e.g., the RNA-dependent RNA polymerase [RdRp] gene region) with the use of low annealing temperatures (as low as 37°C) (18, 19) . This strategy proved to be a promising one, as evidenced by the detection of most of the strains commonly spread in the countries of the European Union, which was confirmed in an international collaborative study (20) .

To increase the sensitivity of RT-PCR detection and still retain a useful amplicon length for sequencing, an RT-booster PCR based on a double amplification performed with the same primer pair was developed (6, 16, 17) .

Strains.

Strain 99-23 372 (GGII, Hu/NLV/Hawaii virus/1971/US) was used to develop and optimize RT-booster PCR . The method was subsequently tested on two additional GGII strains (patient 5 of Turin outbreak in 2002, Hu/NLV/Melksham/1994/UK, and strain 98 39 449, Hu/NLV/Hawaii virus/1971/US) and a GGI strain (patient 41, La Spezia outbreak in 2002, Hu/NLV/Valetta/1995/Malta) .

Sample preparation.

Viral suspensions were prepared as follows . Ten percent stool suspensions in phosphate-buffered saline (Oxoid, Basingstoke, England) from patients implicated in NV outbreaks were clarified by centrifugation at 3,000 rpm for 20 min in an Eppendorf Microfuge . One hundred microliters of the viral suspension of strain 99-23 372 and its 10-fold dilutions (from 10^–1 to 10^–3) were used to experimentally contaminate 900 µl of shellfish extract (final dilution of virus in shellfish ranged from 10^–1 to 10^–4) obtained from samples of Mytilus galloprovincialis prepared as elsewhere described (8) . For the experiments on naturally contaminated shellfish, 75 g of whole shellfish homogenate from 16 samples (Mytilus galloprovincialis and Tapes philippinarum) collected from local markets was processed as described by De Medici et al . (8) and analyzed together with a positive control (shellfish extract contaminated with strain 99-23 372) and negative controls (uninoculated samples) .

RNA extraction.

Both for viral suspensions and experimentally contaminated shellfish, RNA extraction was performed with Nucleospin RNAII (Macherey-Nagel, Düren, Germany) according to manufacturer instructions, and nucleic acid was eluted in a 100-µl volume . For naturally contaminated samples 300 µl of shellfish extract, equivalent to 5 g of shellfish homogenate, was extracted as described by Afzal and Minor (1) and Croci et al . (7) . The RNA pellet was resuspended in 10 µl of deionized triply distilled water containing 1 U of RNase inhibitor/µl .

Primers.

A broadly reactive primer set (JV12 and JV13; synthesized by M-Medical Genenco, Milan, Italy; Table 1), amplifying a 326-bp region of the RdRp gene (18), was chosen for RT-booster PCR and single-round RT-PCR (19) .

 
Optimization of RT-booster PCR.

Tests were performed to define the primer concentrations in the first PCR (1, 10, and 100 nM and 1 µM) and in the second PCR (tenfold higher than the ones used for the first PCR), annealing times (1 min 30 s or 4 min) and number of cycles (20, 30, or 40) in the first PCR, and volume of the first PCR product transferred to the second PCR .

RT-booster PCR and RT-PCR.

RT-booster PCR was performed with the Access RT-PCR system (Promega, Madison, Wis.) . RT was performed with the following mixture: 1x AMV/Tfl buffer, 200 mM deoxynucleoside triphosphates (dNTPs), 1 mM MgSO₄, 5 U of avian myeloblastosis virus reverse transcriptase, 4 µl of 5 µM primer JV13, 5 µl of RNA, and PCR grade water to a final volume of 45 µl . The RT reaction was carried out at 48°C for 60 min (PTC-200 Thermalcycler; MJ Research, Watertown, Mass.) and was terminated with a 5-min step at 99°C . The first PCR was performed by adding 1 µl of Tfl DNA polymerase (5 U/µl), 1 µl of 5 µM primer JV13, 1 µl of 5 µM primer JV12, and 2 µl of PCR grade water directly to the RT vessels . Samples were then denatured for 2 min at 94°C and subjected to 20 cycles of 94°C for 1 min, 37°C for 4 min, and 68°C for 2 min . A final extension at 68°C for 7 min was then performed . For the second PCR, 5 µl of the first PCR product was added to the following mixture: 1x AMV/Tfl buffer (Promega), 200 mM dNTPs, 1 mM MgSO₄, 5 U of Tfl polymerase, 1 µM primers JV13 and JV12, and PCR grade water to a final volume of 50 µl . Thermalcycler conditions were as follows: 94°C for 2 min, followed by 40 cycles at 94°C for 1 min, 37°C for 1 min 30 s, and 68°C for 2 min and a final extension at 68°C for 7 min .

A single-round RT-PCR, performed as described by Vinje and Koopmans (19), was used as a comparative method . Both single-round RT-PCR and RT-booster PCR were repeated three times on RNA extracted from viral suspensions and experimentally contaminated shellfish .

Electrophoresis and Southern blotting.

PCR products were run in 2% agarose gel (Kodak, New Haven, Conn.) in the presence of ethidium bromide and photographed by Chemi Doc (Bio-Rad Laboratories, Hercules, Calif.) . The resulting amplicon (326 bp) was confirmed by Southern blotting; hybridization was performed as described by Bergmans et al . (4) with the probes reported in Table 1 (J . Vinje, personal communication) at a temperature of 42°C .

Data and conclusions.

This paper describes the development of an RT-booster PCR for the detection of NVs both in shellfish and clinical samples . The data obtained showed that the highest sensitivity was reached when the first PCR was performed with a primer concentration of 0.1 µM, while the detection sensitivity decreased at higher (1 µM) and lower concentrations (10 and 1 nM) . The use of a longer annealing time in the first PCR appeared to be slightly more effective in enhancing the overall assay sensitivity than increasing the number of cycles in the first PCR . In fact, a 4-min annealing time instead of 1 min 30 s resulted occasionally in an increase in sensitivity, while an increase in the number of cycles from 20 to 40 didn't produce any change in sensitivity . The use of smaller volumes of the first PCR product in the second PCR resulted in a decrease in the formation of primer dimers, without any evident loss of sensitivity (data not shown) . In the conditions defined the RT-booster PCR was able to detect the NV up to the second decimal dilution beyond the last dilution detected by the single-round RT-PCR (Fig . 1) . The results for the different strains tested consistently showed an increase in sensitivity: for the GGII strains the increase was at least two decimal dilutions, while, in our conditions (no detection in single-round PCR), the increase in sensitivity for the GGI strain, although perceptible as more than two decimal dilutions, could not be clearly defined (Table 2) .

 
The higher sensitivity of booster amplification was confirmed with experimentally contaminated shellfish, and the results were consistent with those obtained with RNA from viral suspensions; RT-booster PCR, in fact, was able to detect NV up to the fourth decimal dilution, while standard RT-PCR detected NV up to the second decimal dilution in all three experiments done (Fig . 2) .

 
On naturally contaminated shellfish RT-booster PCR followed by Southern blotting detected the presence of NVs (confirmed by sequencing GGII NV strains) in 3 out of 16 examined samples, while standard RT-PCR gave negative results on all samples (Fig . 3) . RT-booster PCR gave positive results for one sample in only one of the two experiments (Fig . 3, lane 7), while it gave positive results for two samples in both experiments (Fig . 3, lanes 11 and 13) .

 
As opposed to other published methods that use RT-(semi)nested-PCR-based techniques, the method proposed does not reduce the length of the amplicon produced in the first amplification . This strategy allows the sequencing of a long section of the RdRp gene, a region reported as being a significant one for epidemiological research (9, 13) . In addition, the sequencing of this region or the use of specific probes (19) allows the confirmation of the amplicon, excluding false-positive reactions that can be developed when low-stringency PCR conditions are used, as in this protocol, to detect a target with high genomic variability (10) . The work carried out confirms what was described by Ruano et al . (16): when the target is present in a very low number of copies, the amplification reaction is hampered by the synthesis of primer dimers, with consequent reduction in the target yield . To overcome these problems, the primers must be diluted in such a way as not to exceed the target concentration . Differently from what was proposed by Ruano et al . (16), a longer annealing time only occasionally seemed to raise the PCR detection limit, possibly due to differences in a variety of other PCR parameters (e.g., low annealing temperature) . Finally, it was demonstrated that, in such a double amplification, it is not necessary to use a high number of cycles in the first PCR in order to raise the method's detection limit . This allows the sensitivity of the method to be raised without increasing the time necessary to complete the analysis . Indeed, 3 h more than the time required for a single-round RT-PCR is enough to complete the RT-booster PCR, thus making this method suitable for the investigation and control of public health problems arising from the consumption of shellfish .

ACKNOWLEDGMENTS

We appreciate the support of M . P . G . Koopmans, F . Ruggeri, and M . N . Losio in kindly providing the strains used in this publication .

This work was supported by Sixth Framework of the European Commission project 506359, "Health promoting, safe seafood of high eating quality in a consumer driven fork to farm concept."

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