Establishment And Application Of The Methods For Concentration And Real-time PCR Detection Of Enteric Viruses In Water

More than 140 types of enteric viruses are found in aquatic environments which can cause a wide variety of illnesses in humans such as hepatitis, gastroenteritis, meningitis, fever, rash, conjunctivitis and even diabetes. These viruses are transmitted via the fecal oral route and primarily infect and replicate in the gastrointestinal tract of the host. They are major causes of waterborne and water-related diseases. Extreme examples were the outbreak of 300,000 cases of hepatitis A and 25,000 cases of viral gastroenteritis caused by shellfish harvested from a sewage-polluted estuary in 1988 in Shanghai. In 1991, an outbreak of 79,000 cases of hepatitis E in Kanpur was ascribed to polluted drinking water. There were 173 cases diarrhea reported to be caused by drinking water polluted by norovirus in Dongguan in Guangdong province in 2007. Therfore, more attention should be paid to the viral safety of drinking water by both governments and public because of the effect of viruses on human health.In many countries, including the United States, bacterial indicators such as fecal coliform total coliform and enterococci are adopted to assess the microbiological quality of water. But no significant correlation was shown between levels of bacterial indicators and concentrations of enteric viruses. There were still some enteric viruses detected in drinking water which had met the bacteriological standards. So it is unsafe to completely rely on bacteriological standards to assess the viral quality of any kind of water. To detect the enteric viruses directly can supply more reliable information about viral contamination of water. However the standards of permitted virus number in water and detection method for viruses have not be made and standardized so far because of the difficulty in detection, high cost and workload. The concentration of virus in aquatic environments is usually much lower than that from clinical specimen, and the concentration of a certain virus in clinical specimen could be as 1016 times high as in drinking water. It is difficult to detect the virus in drinking water directly even if using the most sensitive method. Thus the most important precondition is to concentrate the virus from water.The common methods for virus concentration are microporous membrane, diatomaceous filters and glass wool filters. The efficiency varies with different methods. It is necessary to explore an effective, sensitive and convenient method for viral concentrateion. The recovery efficiency of microporous membrane and NanoCeram filter were compared in this study. NanoCeram cartridges, a new electropositive filter with a surface area of 500 to 600 m2/g, can concentrate viruses in water with high turbidity. It is convenient to use the cartridge and the price of it is only 1/5 of 1MDS filter. It is the first time that NanoCeram filter has been used to concentrate viruses in water samples in China.The second concentration of viruses in eluates from washed cartridges is needed because it is still difficult to detect viruses directly after first concentration. Coagulation-precipitation, dialysis and ultracentrifugation are commonly applied for the second concentration. Dialysis and ultracentrifugation are not useful for large volume of first concentrated solution. Flocculants include organic and inorganic chemicals. Three kinds of flocculants were compared in this study and PEG process had the highest efficiency in second concentration among them.After second concentration of the viruses, the volume of the sample solution reduces to small volume similar to clinical specimens which are easy to be assayed with common methods. However, one of huge differences between the environmental and clinical samples for virus test is its diversity. A clinical patient is generally infected with one kind of virus that is readily diagnosed in a laboratory. But for environmental samples, there may be one or much more kinds of viruses in one concentrated sample. Because the susceptibilities of different viruses to their cellular hosts are various, a huge amount of work is required to identify each of them. In addition, there were some kinds of viruses such as norovirus which cannot propagate in cell culture and other kinds of viruses, such as adenovirus serotype 40 and 41 which are difficult to multiply in cells. Molecular biological techniques like gene probe and PCR can overcome these shortages. Although the sensitivity of gene probe is too low to be used, the state of real time PCR has shown many advantages, for example, it is inexpensive, timesaving, and able to detect nonculturable viruses or slow-growing viruses. It is very useful to estimate the public health risks of low levels of enteric viruses in aquatic environments. To perform qPCR, the nucleic acid standards for real time PCR shoud be constructed separately according to different individual virus. The stability and sensitivity of qPCR reaction system should be evaluated. In this study adeno virus serotype 40 and 41, rotavirus group A which are difficult to culture and enterovirus in water samples, were detected by real time PCR Enteric virus were found in many kinds of aquatic environments such as river, lake, sea underground water and even treated drinking water.Chinese investigator detected the enteroviruses in Bohai bay in Tianjing, with the concentration from 6.3×107copies/L tol.7×106copies/L. Rotaviruses were also detected in surface water, raw water, treated drinking water and tap water, with positive rates of 34.6%,11.7%,22.4% respectively in only 2 liters water using RT-nested PCR in China. Twenty years ago, Zhang investigated the presence of enteric viruses in source water of the East Lake and tap water of drinking water treatment plants using cell culture assay in Wuhan. They found that 60% of the raw water samples and 35% of tap water samples were positive for enteric viruses respectively. The average viral removal rate of drinking water treatment plants was only 53.18%. The East Lake as a major drinking water source at that time was polluted severely year by year and was finally abandoned as a potable water resource in the middle of the 1990s. Instead, river water has been the only water resource for drinking water production in the city. However, little has been known about the status of virus pollution in source water and tap water coming from rivers in Wuhan, or in other cities on rivers in China since then.With China's rapid economic development, the Yangtze River and the Hanjiang River have been increasingly polluted by the discharge of untreated or incompletely treated industrial waste, agricultural waste and sewage. By 2007, yearly industrial and urban wastewater released into the Yangtze had exceeded over 30 billion tons,50% of which was untreated. It was reported that there are 14 sewage outfalls near the 18 source-water protection areas, which threatened the quality of source water. The situation of virus in the Yangtze River and the Hangjiang River is still unknown. What is more, little information on viral pollution in any drinking water treatemant plant using both River water as resourses has been reported so far. For the present report, source water and finished water from six water works depending on the Yangtze and the Hanjiang Rivers were collected in Wuhan in four different seasons. Adenovirus, rotavirus and enterovirus were assayed by real time PCR to examine the viral status in the water as well as some other aspects that could impact the real time PCR measurement.Part I Concentration of Virus in Drinking WaterObjective To test and assess the concentration recovery efficiency of viruses with NanoCeram filters, and compare the recovery efficiency of inorganic flocculation with Mg(OH)2, organics flocculation and Polyethylene Glycol (PEG)/NaCl precipitation for the second concentration.Methods For the first concentration:f2 bacteriophage, standing for the enteric virus in water, was spiked to 50L of the source water from the Yangtze River and the Hanjiang River to simulate source water and was spiked to 100L tap water dechlorinated by Na2S2O3 to simulate treated drinking water. The simulated waters were filtered through NanoCeram filters, and then the filters were eluted with 3% beef extract (pH 9.5). After that, the pH of elution solutions were adjusted to 7.0. For the second concentration, f2 phage was spiked into 200ml of 3% beef extract (pH 7.0) to simulate the first concentrated solution. The recovery efficiency of virus by inorganic, organic flocculation and PEG/NaCl precipitation were compared for second concentration. For inorganic flocculant, MgCl2(1M) and K2HPO4(1M) were added to the simulated water and then the pH was adjusted to 8.4 and the recovered precipitate was quantitated subsequently for f2 phage. In the organics flocculantion, pH of the simulatied water was adjusted to 1.5,2.0,3.0,3.5,4.0,5.0, 7.0, and the turbidity of each pH was tested, together with the concentration of f2 phage. The recovery efficiency was compared in different conditions. In PEG/ NaCl system, the recovery efficiency of f2 phage in different concentrations of PEG 8000(4%,6％,8%,10%,12%,13%,14%,16%,18%,20%) was compared. A double agar-layer technique was applied to calculate the concentration of f2 bacteriophage using E coli 285 as its host bacteria.Result The average recovery yield was 50.27%±14.35% for tap water samples and 51.63%±26.60% for source water samples using NanoCeram cartridges for the first concentration. The recovery yield was very low by using inorganic flocculants. Although the turbidity of solution (an indicator of forming flocculate) was getting higher when the pH value was below 3, the recovery yield was still very low(less than 5%) in organic flocculantion. When the concentrations of PEG 8000 were 4% and 6%, the recovery yield was less than 3%, while the concentration reached 8%,the average recovery yield was larger than 70%.The recovery yield was largest (90.09%±10.50%) at the concentration of PEG 8000 being 13%.Conclusion NanoCeram filers combined with PEG/NaCl is an effect method to concentrate virus in drinking water.Part II Construction of Standards for Adenovirus, Rotavirus and Enterovirus for Real Time PCRObjective To constructs nucleic acid standards for HAdV, HAdVF, EV and RVA for real time PCR which were used to quantitate the concentration of each virus.Method The RNA of poliovirus 1 Sabin type and human rotavirus separated from infant feces was extracted using High Pure Viral Nucleic Acid Kit (Roche, Germany), and then reverse-transcribed to cDNA. DNA of adenovirus type 40 was also extracted. The conditions of conventional PCR were optimized with the genomic DNA and cDNA of different viruses by gradient PCR. The templates of each virus were subsequently amplified using the appropriate primers of different viruses in conventional PCR protocol. DNA fragments were recovered from agarose gels, and then were cloned into a pMD 18-Tvector using the TA cloning strategy. The plasmids were transformed into E.coli DH5a and recombinant bacteria were selected on ampicillin-containing LB agar. The positive clones were propagated overnight in liquid culture. The plasmids were extracted from E. coli and the inserts were sequenced to confirm their specificity. Then the sequences were compared to the data posted in GenBank by using the BLAST algorithm. DNA concentration was measured and the copy number was calculated according to the formula. Real time PCR was carried out with the standards of each virus to quantitate the concentration of HAdV, HAdVF, RVA and EV. The stability and sensitivity were assessed. Each real-time PCR mixture (20μl) contained 10μl 2×PCR mix,100 nM each primer, and 2μl DNA template. The amplification procedures included three hold programs, (ⅰ) 2 min. at 50℃, (ⅱ) 10 min. at 95℃, followed by 40 cycles consisting of 15 seconds at 95℃,30seconds at 60℃and 30seconds at 72℃, (ⅲ) then a dissociation curve analysis from 60℃to 95℃. The concentrations of different diluted virus of adenovirus, poliovirus and rotavirus were quantitated by the constructed real time PCR. Meanwhile the relationship between the copy number and dilutions with eachvirus were assessed.Result The best annealing temperature of HAdV, HAdVF, RVA and EV was 60℃in conventional PCR reaction. The courses of connection of specific DNA fragments to pMD 18 plasmid and transformation of the recombinant plasmid to susceptible bacterial cells were successful. The recombinant plasmid included target products. The identities of inserted nucleotide sequences of HAdV, HAdVF, RVA and EV to their general template sequences were 99%(132bp),100%(130bp),100%(161bp), and 99%(143bp), respectively. The concentrations of the final plasmid were 1.2×1011copied/μl (HAdV),3.9x1010copied/μl (HAdVF),9.3×1010copied/μl (EV), and 6.1×010copied/μl (RVA). The amplifying curves with different concentrations of the standards of HAdV, HAdVF, RVA and EV from 10 to 108 copies/μl were separated well and there were only one peak in dissociation curve with each virus with the Tm value of 85.4℃(HAdV),82.6℃(HAdVF),74.8℃(RVA),83.4(EV). The amplifying efficiency were ranged from 89% to 99%.The linear correlations between the copy numbers and titers with different viruses were as follow:R=0.905 for HAdV, R2=0.986 for RVA, R2=0.998 for HAdVF, R2=0.967 for EV.Conclusion The construction of external standards for detection of HAdV, HAdVF, EV and RVA in water using real-time PCR was successful. The established qPCR assay based on recombinant plasmid as standards has good reliability,, repeatability and sensitivity to quantify HAdV, HAdVF, EV and RVA in water. As few as 10 copies can be reliably detected using the standards of HAdV, HAdVF, EV and RVA by real-time PCR. There are close linear correlation between the copy numbers and titers for each virus.PartⅢDetection of Adenovirus, Rota virus and Entero virus in Source Water and Treated Drinking WaterObjective To quantify the concentration of total human adenovirus(HAdV), human adenovirus group F(HAdVF), human rotavirus group A(RVA) and enterovirus(EV) in source water and treated drinking water in four seasons in 2011, by using the constructed method of concentration in part 1 and the method of real time PCR in part 2. To evaluate the viral removal efficiency of water treatment processes in drinking water treatment plants and its correlation with different physicochemical and biological parameters.Method Water samples(1001 for source water,2001 for finished water) were collected from the six drinking water treatment plants (three drinking water treatment plants are situated on the banks of the Yangtze River, and the other three plants on the banks of the Hanjiang River) in four seasons separately:winter (Jan.2011); spring (Apr.2011); summer (Jul.2011) and autumn (Oct.2011). In order to obtain more information about the water characteristics, several physicochemical parameters such as turbidity, temperature, pH, and residual chlorine as well as fecal coliform were measured. Finished water (2001) dechlorinated by adding 50 mg of sodium thiosulfate per liter and source water (1001) were filtered through NanoCeram VS2.5-5 virus filters. Filters were changed for each sample and used only once. The cartridges of the samples were transported to the laboratory in ice box after collection and were subsequently eluted by 600m1 3% beef extract (pH 9.5). PEG/NaCl precipitation was used for the second concentration. PEG8000 (13%) and NaCl (1.2%) were added to the elution solution adjusted the pH to 7.0. Then the solution was centrifugated at 10000g for 30min. The pellet was finally suspended in 10 ml PBS (pH 7.0). The nucleic acids of water samples were extracted. RNA was reverse-transcribed for cDNA synthesis. Real-time PCR was performed by SYBR Green I fluorescent dye strategy to quantify the concentration of HAdV, HAdVF, RVA and EV in water samples. The viral removal efficiency of water treatment process in drinking water treatment plants was calculated and the correlations with different parameters were analyzed by Pearson correation. The PCR inhibition effects in source water and treated water were comparably evaluated by adding a given amount of standard nucleic acids and two ways, centrifugation and dilution, of removal of those PCR inhibitors were adopted.Result HAdV, HAdVF and RVA were all positively detected in the samples of source water and treated drinking water. EV could be found in 46%(11/24) of all the source water samples, but only 21%(5/24) positive in treated drinking water. The concentration of enteric viruses in source water of the Yangtze River were as follow: 5.54×103copies/L～2.20x 106copies/L (HAdV); 5.69×104copies/L-3.76×105copies/L(HAdVF) ;6.91×103copies/L～1.11×105copies/L (RVA); 0～7.95×103copies/L(EV). The concentration of enteric viruses in source water of the Hanjiang River were as follow 2.75×103copies/L～1.05x106copies/L(HAdV); 5.66×102copies/L～4.81×104copies/L(HAdVF); 0～5.56×103copies/L(EV); 4.84×103copies/L～4.52×104copies/L(RVA). The highest removal rate was EV (97%), followed by RVA (82%), HAdV (73%) and HAdVF (72%). In our study, about 57.04% and 61.20% of the spiked DNA could be detected in the nucleic acids of source water samples, while 96.46% and 91.07% of them could be detected in treated drinking water, indicating that there were some factors influencing the underestimating of the viral concentrations of water samples. The increased rates of recovery via seeding HAdV standards ranged from 29.7% to 198.2% in various sets of nucleic acid extraction (t=10.235, P=0.000). However no improvement was observed for RVA in source water samples after centrifugation (t=1.027, P=0.338). There were significant correlations between the concentration of HAdV and the turbidity of the Yangtze River (R2=0.768, p=0.004), between HAdVF and the turbidity of the Hanjiang (R2=0.711, P=0.010). No correlation between fecal-indicator bacteria and enteric viruses was observed. The concentration of rotavirus in the Yangtze River was higher than that in the Hanjiang River (P =0.035).Conclusion Although the parameters of pH, turbidity, fecal coliforms and residual chlorine have all met the national standards for drinking water, a large number of HAdV, HAdVF, RVA and EV in source water and finished water have been measured by qPCR in six water works in Wuhan. Centrifugation and dilution are good ways to get rid of PCR inhibitor to increase the accuracy of qPCR assays.