Hepatitis A (live) vaccine (attenuated HAV H2 strain) was purchased from the Institute of Medical Biology, Chinese Academy of Medical Sciences, China, and stored at 4 ℃ until use.
Tap water was collected from the laboratory in the Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), China. Donghu Lake water samples were collected from Donghu Lake, Wuhan, China.
RNA extraction and reverse transcription (RT) of virus RNA were performed according to the instructions supplied with the RNA extraction kit (Takara) and mouse reverse transcription kit (MLV, Promega), respectively. The amount of HAV RNA used for reverse transcription was 125 ng. HAV gene fragments were amplified by polymerase chain reaction (PCR) using a Phusion High-Fidelity PCR kit (New England BioLabs). The primers used to amplify the genes are shown in Supplementary Table S1 (Cohen et al., 1987; Brooks et al., 2005). The positive plasmid pGEM-VP1 was constructed and considered the standard plasmid. After optimizing annealing temperature and reaction conditions, HAV-F2 and HAV-R2 were selected for subsequent experiments (Supplementary Figure S1).
The density of the plasmid was measured with a Nano-Drop 2000 (Thermo Fisher Scientific, Inc.) and the re-sult was converted into the viral copy number. The plasmid used for the standard curve was packed and stored at -20 ℃.
The quantitative detection of HAV was performed by SYBR Green real-time PCR. The pGEM-VP1 plasmids were 10-fold serial-diluted, and solutions of 101 copies/μL, 102 copies/μL, 103 copies/μL, 104 copies/μL, 105 copies/μL, 106 copies/μL, 107 copies/μL, and 108 copies/μL were used to draw a standard curve, with three replicates per dilution. SYBR Green real-time PCR was carried out in a Bio-Rad CFX Manager instrument (Bio-Rad) in a 20-μL reaction volume that contained 1 μL cDNA template, 0.5 μL each of forward and reverse primers, 10-μL 2×SsoFast EvaGreen Supermix (Bio-Rad), and double-distilled water (ddH2O). The thermal profile for SYBR Green real-time PCR was 95 ℃ for 3 min, followed by 40 cycles of 95 ℃ for 10 s and 55 ℃ for 30 s.
Sample collection containers and viral concentration containers used in all steps were sterilized and each experiment had three replicates. The different concentration methods are shown in Figure 1.
HAV (1 mL of 5.66×106 copies/μL) was diluted in 500 mL tap water, and 5 mL of 0.4% sodium thiosulfate (Na2S2O3) was added to remove hypochlorous acid. The anode membrane (Immobilon-Ny+) (47-mm diameter, 0.45-μm pore size, Millipore INYC00010) was used with negative pressure suction to adsorb the virus.
For method A, the anode membrane was moved to a dish (diameter 60 mm), and 2 mL Trizol added to soak this membrane for 5-10 min, after which the Trizol ly-sate was collected and RNA was extracted.
For method B, after filtering, the filtration membrane was transferred to a 30-mL solution composed of 13% polyethylene glycol (PEG)6000 and 0.3 mol/L NaCl buffer (pH 7.0), then mixed thoroughly at 4 ℃ overnight (Jaykus et al., 1996). The viruses were collected by centri-fugation at 13, 000×g for 1 h, the supernatant was gently discarded, and 2 mL Trizol was added to extract RNA.
For method C, the filtration membrane was transferred to 30 mL of 0.01 mol/L EDTA -0.1 mol/L glycine buffer (pH 10.5), which contained 0.1% bovine serum albumin (BSA), for 1 h. Solid PEG 6000 and NaCl were added to the elutes to attain final concentrations of 13% and 0.3 mol/L, respectively, then stirred well and shaken at 4 ℃ for 8 h. The viruses were collected by centrifugation at 13, 000 × g for 1 h, then the supernatant was gently discarded and 2 mL Trizol was added to extract RNA.
HAV (1 mL of 5.66×106 copies/μL) was added to 500 mL tap water or PBS, and 0.4% sodium thiosulfate (Na2S2O3) solution was added to the tap water to remove hypochlorous acid. First, the tap water and PBS solution were filtered through a pre-filtration membrane (glass cellulose membrane, 2.7-μm pore size). Then the HAV was concentrated through an Immobilon-Ny+ filtrating membrane. The concentration efficiency with and without pre-filtration were compared to determine the impact of pre-filtering on concentration efficiency.
Six 500-mL PBS samples containing 1 mL of 5.66×106 copies/μL HAV were concentrated through six different membranes with different pore sizes and materials (A, F, G, H, I, and J) (Table 1). Then, 2 mL Trizol was added to the filtration membrane and allowed to soak for 5-10 min prior to RNA extraction.
Table 1. Different filter membranes used in the present study
We collected 9 water samples (500-mL for each sample) from Donghu Lake. The Donghu Lake water sample containing 1 mL of 5.66×106 copies/μL HAV was defined as group 1. Additional 500-mL Donghu Lake water samples were mixed with components of PBS (i.e., with NaCl, KCl, Na2HPO4, and KH2PO4) to final concentrations of 137 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L Na2HPO4, and 2 mmol/L KH2PO4, respectively. The lake water sample containing components of PBS was spiked with HAV and defined as group 2. The Donghu Lake water sample including components of PBS was defined as the negative control group. The water samples from group 2 and the negative control group were adjusted to pH 7.2-7.4 with HCl. The water samples were all centrifuged at 4 ℃, 3800×g, for 15 min, to remove large particles. The supernatant was filtered through a pre-filtration membrane and a mixed cellulose ester (MCE) membrane. After filtration, 2 mL Trizol was added to the MCE membrane and it was soaked 5-10 min prior to RNA extraction.
To determine whether inhibitors in the water might affect SYBR Green real-time PCR amplification, the samples were treated as follows. First, cDNA (4.5 μL) from the water sample mixed with 0.5 μL ddH2O was defined as A1; 4.5 μL cDNA from the water sample mixed with 0.5 μL standard plasmid (B stands for stan-dard plasmid, of which there were 3.5×105 copies) was defined as A2; 1 μL of the A1 or A2 template was used in SYBR Green real-time PCR. Next, cDNA (5 μL) from the water sample was mixed with 45 μL ddH2O. A 4.5-μL sample of the resulting solution mixed with 0.5 μL ddH2O was defined as A3, and a 4.5-μL sample of the solution mixed with 0.5 μL standard plasmid was defined as A4; 1 μL of the A3 or A4 template was used in SYBR Green real-time PCR.
When HAV was concentrated from tap water samples and PBS samples, virus recovery was calculated using the following formula:
When HAV was concentrated from Donghu Lake water samples, virus recovery was calculated using the following formula:
The results of the test groups were evaluated by Student's t-test; if the P-value was less than 0.05, the difference was considered significant.
Hepatitis A virus (HAV)
RNA extraction and construction of HAV standard plasmid
HAV quantitative detection by SYBR Green real-time RT-PCR
Different concentration methods
Improving recovery efficiency by pre-filtration
Different kinds of filtration membranes
Processing of water samples from Donghu Lake
Virus recovery efficiency
To exclude the interference of impurities in natural water bodies, tap water was chosen to establish a labora-tory method of enriching HAV. HAV (1 mL of 5.66×106 copies/μL) was added to 500 mL tap water. Virus concentration was performed using the anode membrane, or the anode membrane combined with secondary elution methods. Copy numbers of concentrated HAV were measured with real-time RT-PCR. As shown in Figure 2A, for tap water, the recovery rate of method A was significantly higher than the recovery rates of methods B and C (P < 0.01) (Figure 2A). The HAV concentration efficiency using anode membrane with no elution is significantly higher than when using anode membrane combined with secondary elution methods.
Figure 2. Recovery efficiency using different concentration methods. Hepatitis A virus (HAV) (1 mL of 5.66×106 copies/μL) was diluted in 500 mL tap water. The HAV in water samples was concentrated by methods A, B, and C, as described in the Materials and Methods section. **, significant difference (P < 0.01). Values represent the mean ± SD of each group.
Impurities in tap water may affect the results of RT-qPCR and lead to a low concentration efficiency. A 2.7-μm pore size glass cellulose membrane was therefore used to remove large particles. For tap water, the concentration efficiency was significantly higher with pre-filtering (P < 0.01) (Figure 2B).
To verify whether salinity could influence the reco-very rate of HAV, PBS was added to the tap water, producing the following concentrations: NaCl 137 mmol/L, KCl 2.7 mmol/L, Na2HPO4 10 mmol/L, and KH2PO4 2 mmol/L. HAV was resuspended in the tap water and PBS buffer mixture, hereinafter referred to as the water sample and the PBS sample. The recovery rate was significantly higher for samples that contained PBS (P < 0.01) (Figure 2B).
To verify whether the size and type of membrane pore could affect concentration efficiency, six types of filtration membrane were used to concentrate HAV in PBS samples: 0.1-μm and 0.2-μm MCE membrane (methods F and G); 0.1-μm and 0.2-μm polyvinylidene fluoride (PVDF) membrane (methods H and I); 0.45-μm anode membrane (method A); and 0.2-μm nylon membrane (method J). As shown in Figure 2C, the virus recovery rate was highest after the use of method F, reaching 92.62 ± 5.17%. Method A had the next highest rate, although it was significantly lower (P < 0.05), and method Ⅰ had the lowest rate, only 1.19 ± 0.38%. The recovery rates of 0.1-μm and 0.2-μm MCE membrane and anode membrane were all higher than 50%(Figure 2C).
The optimal concentration method, as described above, was applied to the Donghu Lake samples. The highest concentration efficiency of HAV detected in raw Donghu Lake samples was 204 copies/μL and the Cq (quantification cycle) value was 35.68 (Table 2). The virus recovery rate for group 2 (containing PBS) was higher than that for group 1 (with no PBS). The use of optimum concentration methods for the Donghu Lake samples, including adding PBS, using pre-treatment after centrifugation, pre-filtering, using MCE membrane filtration, and not performing elution, provided a virus recovery rate of 79.45 ± 9.88%.
Table 2. Recovery efficiency of hepatitis A virus (HAV) of water samples in Donghu Lake
As described for groups A1, A2, A3, A4, and B in the Materials and Methods section, the plasmid copy number of each group was calculated as follows to determine whether dilution could influence the inhibitor:
Recovery rate with no dilution = A2/(A1+B)
Recovery rate with 1:10 dilution = A4/(A3+B)
Three groups of parallel tests were performed. The virus recovery rate increased by 19.73%-26.74% when the sample was diluted 10 times (Table 3). Therefore, dilution treatment could partially remove the inhibitory factor for HAV detection.
Table 3. The virus recovery efficiency after PCR inhibitor treatment
Product Primer name Primer sequence (5'-3') HAV247bp HAV-Fa GTTTTGCTCCTCTTTATCATGCTATG HAV-Ra GGAAATGTCTCAGGTACTTTCTTTG HAV HAV-F2 TGCTATGGATGTTACTAC HAV-R2 ATCTTTCATGGTTGTTATAC HCV HCV-F TCT GCG GAA CCG GTG AGT A HCV-R TCA GGC AGT ACC ACA AGG C AIV AIV-F TAT GAG AAG TGA AGT GGA A AIV-R GTG TAT GTT GTG GAA TGG HIV HIV-F TGT GTG CCC GTC TGT TGT GT HIV-R GAG TCC TGC GTC GAG AGA GC Note: a The primer HAV-F/R was used according to Brooks (2005).
Table S1. The primers used for hepatitis A virus (HAV) detection with conventional and real-time RT-PCR