水体病毒钙离子絮凝浓缩新方法研究

研究建立了一种从水体中浓缩病毒的新方法,即钙离子絮凝-柠檬酸缓冲液洗溶法,该方法的要点是先用一定量的钙离子溶液和钙离子絮凝剂絮凝水体中的病毒,再用pH5.0的0.3mol/L的柠檬酸缓冲液洗溶,然后再进一步超滤浓缩。此法可方便地将水体中的病毒浓缩10000倍以上。应用该法分别对人工播种于饮用水中的f2噬菌体和脊髓灰质炎疫苗病毒(PV1)进行了浓缩,结果发现f2噬菌体的平均回收率达96%,而PV1的回收率为100%,均显著高于阳电膜过滤法(P<0.05)。该方法快速、简便、有效。     

Detection of noroviruses in tap water in Japan by means of a new method for concentrating enteric viruses in large volumes of freshwater

A virus concentration method using a cation-coated filter was developed for large-volume freshwater applications. Poliovirus type 1 (LSc 2ab Sabin strain) inoculated into 40 ml of MilliQ (ultrapure) water was adsorbed effectively to a negatively charged filter (Millipore HA, 0.45- micro m pore size) coated with aluminum ions, 99% (range, 81 to 114%) of which were recovered by elution with 1.0 mM NaOH (pH 10.8) following an acid rinse with 0.5 mM H(2)SO(4) (pH 3.0). More than 80% poliovirus recovery yields were obtained from 500-ml, 1,000-ml, and 10-liter MilliQ water samples and from tap water samples. This method, followed by TaqMan PCR detection, was applied to determine the presence of noroviruses in tap water in Tokyo, Japan. In a 14-month survey, 4 (4.1%) and 7 (7.1%) of 98 tap water samples (100 to 532 liters) contained a detectable amount of noroviruses of genotype 1 and genotype 2, respectively. This method was proved to be useful for surveying the occurrence of enteric viruses, including noroviruses, in large volumes of freshwater.     

Detection of Noroviruses in Tap Water in Japan by Means of a New Method for Concentrating Enteric Viruses in Large Volumes of Freshwater

A virus concentration method using a cation-coated filter was developed for large-volume freshwater applications. Poliovirus type 1 (LSc 2ab Sabin strain) inoculated into 40 ml of MilliQ (ultrapure) water was adsorbed effectively to a negatively charged filter (Millipore HA, 0.45-μm pore size) coated with aluminum ions, 99% (range, 81 to 114%) of which were recovered by elution with 1.0 mM NaOH (pH 10.8) following an acid rinse with 0.5 mM H₂SO₄ (pH 3.0). More than 80% poliovirus recovery yields were obtained from 500-ml, 1,000-ml, and 10-liter MilliQ water samples and from tap water samples. This method, followed by TaqMan PCR detection, was applied to determine the presence of noroviruses in tap water in Tokyo, Japan. In a 14-month survey, 4 (4.1%) and 7 (7.1%) of 98 tap water samples (100 to 532 liters) contained a detectable amount of noroviruses of genotype 1 and genotype 2, respectively. This method was proved to be useful for surveying the occurrence of enteric viruses, including noroviruses, in large volumes of freshwater.     

Structural basis for norovirus inhibition and fucose mimicry by citrate

Human noroviruses bind with their capsid-protruding domains to histo-blood-group antigens (HBGAs), an interaction thought to direct their entry into cells. Although human noroviruses are the major cause of gastroenteritis outbreaks, development of antivirals has been lacking, mainly because human noroviruses cannot be cultivated. Here we use X-ray crystallography and saturation transfer difference nuclear magnetic resonance (STD NMR) to analyze the interaction of citrate with genogroup II (GII) noroviruses. Crystals of citrate in complex with the protruding domain from norovirus GII.10 Vietnam026 diffracted to 1.4 Å and showed a single citrate bound at the site of HBGA interaction. The citrate interaction was coordinated with a set of capsid interactions almost identical to that involved in recognizing the terminal HBGA fucose, the saccharide which forms the primary conserved interaction between HBGAs and GII noroviruses. Citrate and a water molecule formed a ring-like structure that mimicked the pyranoside ring of fucose. STD NMR showed the protruding domain to have weak affinity for citrate (460 μM). This affinity, however, was similar to the affinities of the protruding domain for fucose (460 μM) and H type 2 trisaccharide (390 μM), an HBGA shown previously to be specifically recognized by human noroviruses. Importantly, competition STD NMR showed that citrate could compete with HBGA for norovirus binding. Together, the results suggest that citrate and other glycomimetics have the potential to block human noroviruses from binding to HBGAs.     

Detection of virus in water: sensitivity of the tentative standard method for drinking water.

The sensitivity of several microporous virus-adsorbent media for reliably detecting low levels of poliovirus from 380 and 1,900 liters of drinking water by use of the tentative standard method was investigated. The virus-adsorbent media tested were (i) nitrocellulose membrane filters, (ii) epoxy-fiber glass-asbestos filters, (iii) yarn-wound fiber glass depth filters, and (iv) epoxy-fiber glass filter tubes. Virus was adsorbed to the filter media at pH 3.5 and eluted with glycine buffer, pH 11.5. The results from 44 samples demonstrated that poliovirus was detected with a 95% reliability at mean virus input levels of 3 to 7 plaque-forming units/380 liters when 1,900 liters of water was sampled. At mean virus input levels of less than 1 to 2 plaque-forming units/380 liters, the detection reliability was 66% in 76 samples when 1,900 liters of water was sampled. No significant difference in virus detection sensitivity was observed among the various virus adsorbent media tested. Overall virus recovery efficiency ranged from 28 to 42%, with a grand average of 35%. Members of the coxsackievirus groups A and B, echovirus, and adenovirus were also detected when 380 and 1,900 liters of water were sampled. These experimental observations attest to the sensitivity of the tentative standard method for detecting low levels of virus in large volumes of drinking water.     

Detection of virus in water: sensitivity of the tentative standard method for drinking water.

The sensitivity of several microporous virus-adsorbent media for reliably detecting low levels of poliovirus from 380 and 1,900 liters of drinking water by use of the tentative standard method was investigated. The virus-adsorbent media tested were (i) nitrocellulose membrane filters, (ii) epoxy-fiber glass-asbestos filters, (iii) yarn-wound fiber glass depth filters, and (iv) epoxy-fiber glass filter tubes. Virus was adsorbed to the filter media at pH 3.5 and eluted with glycine buffer, pH 11.5. The results from 44 samples demonstrated that poliovirus was detected with a 95% reliability at mean virus input levels of 3 to 7 plaque-forming units/380 liters when 1,900 liters of water was sampled. At mean virus input levels of less than 1 to 2 plaque-forming units/380 liters, the detection reliability was 66% in 76 samples when 1,900 liters of water was sampled. No significant difference in virus detection sensitivity was observed among the various virus adsorbent media tested. Overall virus recovery efficiency ranged from 28 to 42%, with a grand average of 35%. Members of the coxsackievirus groups A and B, echovirus, and adenovirus were also detected when 380 and 1,900 liters of water were sampled. These experimental observations attest to the sensitivity of the tentative standard method for detecting low levels of virus in large volumes of drinking water.     

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.     

Detection of cryptosporidia and Cryptosporidium parvum oocysts in environmental water samples by immunomagnetic separation-polymerase chain reaction

Cryptosporidium parvum has emerged as one of the most important new contaminants found in drinking water. Current protocols for the detection of cryptosporidia are time-consuming and rather inefficient. We recently described an immunomagnetic separation-polymerase chain reaction (IMS-PCR) assay permitting highly sensitive detection of C. parvum oocysts in drinking water samples. In this study, a second IMS-PCR assay to detect all cryptosporidial oocysts was developed, and both IMS-PCR assays were optimized on river water samples. A comparative study of the two IMS-PCR assays and the classical detection method based on an immunofluorescence assay (IFA) was carried out on 50 environmental samples. Whatever the type of water sample, the discrepancy in C. parvum detection between the IFA and IMS-PCR took the form of IFA-negative/IMS-PCR-positive results, and was caused mainly by the greater sensitivity of IMS-PCR as compared with IFA. Of the 50 water samples, only five tested positive for C. parvum using IMS-PCR, and could constitute a threat to human health. These results show that both IMS-PCR assays provide a rapid (1 d) and sensitive means of screening environmental water samples for the presence of cryptosporidia and C. parvum oocysts.     

枸橼酸转运蛋白mRNA在代谢性酸中毒大鼠肾组织的表达

文章报道了代谢性酸中毒时大鼠肾组织两种钠离子依赖的枸橼酸膜转运蛋白ｍＲＮＡ表达量的变化。给雌性Ｗｉｓｔａｒ大鼠喂含０．２８ｍｏｌ／Ｌ ＮＨ４Ｃｌ饮用水诱导产生代谢性酸中毒。喂酸后分别于１、３、７ｄ处死大鼠,测定血浆ＨＣＯ＾－３浓度的变化。以Ｎｏｒｔｈｅｒｎ杂交方法,用钠离子依赖的枸橼酸膜转运蛋白１（ＳＤＣＴ１）探针及钠离子依赖的枸橼酸膜转运蛋白２（ＳＤＣＴ２）探针,分别检测肾皮质枸橼酸转运蛋白     

Detection and discrimination of Cryptosporidium parvum and C. hominis in water samples by immunomagnetic separation-PCR

Cryptosporidium parvum and C. hominis have been the cause of large and serious outbreaks of waterborne cryptosporidiosis. A specific and sensitive recovery-detection method is required for control of this pathogen in drinking water. In the present study, nested PCR-restriction fragment length polymorphism (RFLP), which targets the divergent Cpgp40/15 gene, was developed. This nested PCR detected only the gene derived from C. parvum and C. hominis strains, and RFLP was able to discriminate between the PCR products from C. parvum and C. hominis. To evaluate the sensitivity of nested PCR, C. parvum oocysts inoculated in water samples of two different turbidities were recovered by immunomagnetic separation (IMS) and detected by nested PCR and fluorescent antibody assay (FA). Genetic detection by nested PCR and oocyst number confirmed by FA were compared, and the results suggested that detection by nested PCR depends on the confirmed oocyst number and that nested PCR in combination with IMS has the ability to detect a single oocyst in a water sample. We applied an agitation procedure with river water solids to which oocysts were added to evaluate the recovery and detection by the procedure in environmental samples and found some decrease in the rate of detection by IMS.     

Detection of viruses in drinking water by concentration on magnetic iron oxide.

Discharge of raw domestic wastes containing human enteric viruses into water courses, consumption of untreated water from canals, streams, and shallow wells in villages, and cross-contamination of water in the distribution system because of intermittent water supply in urban areas continue to cause widespread outbreaks of infectious hepatitis in India. To detect a low number of viruses in 50- to 100-liter samples of water, a method was developed with magnetic iron oxide as the virus adsorbent. Poliovirus-seeded dechlorinated tap water, adjusted to pH 3.0 and 0.0005 M AlCl3, was filtered through a 10-g bed of iron oxide sandwiched between two AP20 prefilter pads held in a 142-mm-diameter, stainless-steel holder. Virus was eluted from iron oxide by recirculating three times a 100-ml volume of 3% beef extract, pH 9.0. The eluate was reconcentrated to 5 ml by adjusting to pH 3, adding 1 g of iron oxide, stirring for 30 min, and eluting the readsorbed virus with 5 ml of beef extract, pH 9.0. Virus recovery varied from 60 to 80%. Using the above method, we took a survey of drinking water at three locations in Nagpur during 1976 and found the presence of virus in 7 of 50 samples. The quantity of virus recovered ranged from 1 to 7 plaque-forming units per 30 to 60 liters. Virus was detected in some samples even with residual chlorine. No coliforms were detected in the virus-positive samples.     

Detection of adenoviruses and enteroviruses in polluted waters by nested PCR amplification.

A procedure has been developed for the rapid detection of enteroviruses and adenoviruses in environmental samples. Several systems for virus concentration and extraction of nucleic acid were tested by adding adenovirus type 2 and poliovirus type 1 to different sewage samples. The most promising method for virus recovery involved the concentration of viruses by centrifugation and elution of the virus pellets by treatment with 0.25 N glycine buffer, pH 9.5. Nucleic acid extraction by adsorption of RNA and DNA to silica particles was the most efficient. One aliquot of the extracted nucleic acids was used for a nested two-step PCR, with specific primers for all adenoviruses; and another aliquot was used to synthesize cDNA for a nested two-step PCR with specific primers for further detection of seeded polioviruses or all enteroviruses in the river water and sewage samples. The specificity and sensitivity were evaluated, and 24 different enterovirus strains and the 47 human adenovirus serotypes were recognized by the primers used. The sensitivity was estimated to be between 1 and 10 virus particles for each of the species tested. Twenty-five samples of sewage and polluted river water were analyzed and showed a much higher number of positive isolates by nested PCR than by tissue culture analysis. The PCR-based detection of enteroviruses and adenoviruses shows good results as an indicator of possible viral contamination in environmental wastewater.     

Development of a concentration method for detection of tobacco mosaic virus in irrigation water

Tobacco mosaic virus (TMV) causes significant yield loss in susceptible crops irrigated with contaminated water. However, detection of TMV in water is difficult owing to extremely low concentrations of the virus. Here, we developed a simple method for the detection and quantification of TMV in irrigation water. TMV was reliably detected at concentrations as low as 10 viral copies/μL with real-time PCR. The sensitivity of detection was further improved using polyethylene glycol 6000 (PEG6000, MW 6000) to concentrate TMV from water samples. Among the 28 samples from Shaanxi Province examined with our method, 17 were tested positive after virus concentration. Infectivity of TMV in the original water sample as well as after concentration was confirmed using PCR. The limiting concentration of TMV in water to re-infect plants was determined as 10² viral copies/mL. The method developed in this study offers a novel approach to detect TMV in irrigation water, and may provide an effective tool to control crop infection.     

Detection of toxigenic Vibrio cholerae from environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure

A pit-stop semi-nested PCR assay for the detection of toxigenic Vibrio cholerae in environmental water samples was developed and its performance evaluated. The PCR technique amplifies sequences within the cholera toxin operon specific for toxigenic V. cholerae. The PCR procedure coupled with an enrichment culture detected as few as four V. cholerae organisms in pure culture. Treated sewage, surface, ground and drinking water samples were seeded with V. cholerae and following enrichment, a detection limit of as few as 1 V. cholerae cfu ml(-1) was obtained with amplification reactions from crude bacterial lysates. The proposed method, which includes a combination of enrichment, rapid sample preparation and a pit-stop semi-nested PCR, could be applicable in the rapid detection of toxigenic V. cholerae in environmental water samples.     

Free amino acid determination in whole peanut seeds

A method which circumvents the lengthy procedural steps for sample preparation in the analysis of free amino acids (FAA) in the peanut is described. Sample preparation consists of extracting a full fat meal with a lithium citrate sample buffer for 15 min. The sample is centrifuged and filtered with two Millipore filters in series. The resulting extract is analyzed "as is" for FAA via a physiological amino acid procedure. The method significantly reduces sample preparation time and eliminates the use of hazardous chemicals. It is well adapted to the analysis of large numbers of samples or samples of limited quantity. It should be applicable to the analysis of FAA in other oilseeds.