माइक्रोबियल स्रोत विशिष्ट मानव और पशु वायरस का उपयोग ट्रैकिंग के लिए लागत प्रभावी विधि
Summary
अध्ययन मल / मूत्र या पानी में नाइट्रेट मानव / शूकरीय / गोजातीय डीएनए वायरस, एडिनोवायरस और polyomaviruses, MST उपकरण के रूप में प्रस्तावित की विशिष्ट मात्रा का ठहराव के लिए qPCR का उपयोग करके संदूषण संदूषण के स्रोत की पहचान के लिए एक लागत प्रभावी विधि का वर्णन करता है.
Abstract
Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.
Protocol
Discussion
प्रतिलिपि प्रस्तुत करने योग्य, विश्वसनीय, सरल, और लागत प्रभावी: वर्णित प्रक्रिया दिनचर्या पर्यावरण और सार्वजनिक स्वास्थ्य प्रयोगशालाओं के लिए एक उपयुक्त विधि के लिए शर्तों को पूरा होगा. प्रोटोकॉल…
Disclosures
The authors have nothing to disclose.
Acknowledgements
इस काम आंशिक रूप से स्पेनिश सरकार "Ministerio डे Educación y Ciencia" (परियोजना AGL2008-05275-C01/ALI) द्वारा समर्थित किया गया था, यूरोपीय संघ अनुसंधान 7 फ्रेमवर्क वित्त पोषित परियोजनाओं VIROBATHE (अनुबंध सं 513648), (VIROCLIME अनुबंध सं 243923, ) और पानी के कातालान एजेंसी, AGENCIA Catalana de l'(एसीए) Aigua, Departament डे नियंत्रण मैं Millora dels Ecosistemes तैराकी द्वारा. विकसित अध्ययन के दौरान मार्ता Rusiñol कातालान सरकार "AGAUR" (FI DGR से) के एक साथी था.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| High speed centrifuge (8,000xg) | Berckman Coulter | Avanti J-20XP | |
| pH-meter, thermometer and conductimeter | Afora | LPPC3003 | |
| Plastic tubes 100-200 cm length | Deltalab | 350059 | |
| Sterile graduated disposable pipettes | Labclinics | PN10E1 | |
| Sterile plastic tubes of 1.5 and 10-15 mL (Eppendorf, Falcons, etc.) | Afora | KA298/00 | |
| Centrifuge pots (500 mL) | Fisher Scientific | SE5753512 | |
| Magnetic stirrers and magnets (one per sample) | Fisher Scientific | 10510 | |
| Glass or plastic containers having flat bottoms to allow the use of magnetic stirrers | Deltalab | 191642 | |
| A peristaltic pump for removing the supernatant (or a water-jet vacuum pump) | Watson-Marlow | 323E/D | |
| Timer to switch-off the stirring after 8-10 hours | Deltalab | 900400 | |
| Hydrochloric acid (1N and 0.1N) | Panreac | 141020.1611 | |
| Sodium hydroxide (1N) | Panreac | 131687.1211 | |
| Artificial seawater sea salts | Sigma | S9883 | |
| Skimmed milk (SM) | Difco | 232100 | |
| Phosphate buffer pH 7,5 | 1:2 v/v of sterile Na2HPO4 0,2M and NaH2PO4 0,2M at pH 7.5 | ||
| Thiosulphate | Panreac | 121879.1209 | Make a 10% solution in water |
| QIAamp Viral RNA Mini Kit | Qiagen | 52904 | |
| 96-well optical reaction plate (500 units) | Applied Biosystems | 43426659 | |
| Optical adhesive covers (100 units) | Applied Biosystems | 4311971 | |
| TaqMan Environmental PCR Master Mix 2x | Applied Biosystems | 4396838 |
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