और, TaqMan करके Genogrouping बच्चों के मल से noroviruses डिटेक्शन एक कदम RT-पीसीआर
Summary
एक कदम और बच्चों के दस्त, कि प्राइमरों और TaqMan खुला पढ़ने फ्रेम 1 है (ORF1) ORF2 जंक्शन क्षेत्र, Norovirus जीनोम का सबसे संरक्षित क्षेत्र है करने के लिए विशिष्ट जांच इस्तेमाल से में Norovirus आइसोलेट्स की genogroup पहचान का पता लगाने के लिए परख RT-पीसीआर वर्णित. एक गैर वाणिज्यिक, लागत प्रभावी शाही सेना निष्कर्षण विधि की विस्तृत.
Abstract
Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. 1, 2, 3
For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer.
Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment 1, 2, 3. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method4 which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate.
For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. 5 is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.
Protocol
Discussion
का प्रयोग विधि – घर में मल के नमूने से न्यूक्लिक एसिड अलग करने के लिए आर्थिक, हम वाणिज्यिक QIAmp क्विएज़न से वायरल शाही सेना किट के साथ के रूप में समान परिणाम प्राप्त करने के लिए, और एक साथ TaqMan RT-पीसीआर हमारी प्…
Disclosures
The authors have nothing to disclose.
Acknowledgements
लेखकों के लिए नवम्बर के लिए एक मानक और नियंत्रण सकारात्मक की तरह उपहार के लिए रोग नियंत्रण और रोकथाम के लिए राष्ट्रीय Calicivirus प्रयोगशाला केंद्र (सीडीसी) का शुक्रिया अदा करना होता है, और जॉन्स हॉपकिन्स पर अभिकर्मकों प्रदान करने के लिए सार्वजनिक स्वास्थ्य के स्कूल की प्रयोगशालाओं.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Guanidine isothiocyanate | Sigma-Adrich | G9277 | |
| Tris HCL | Sigma-Aldrich | T5941 | |
| EDTA | Sigma-Aldrich | E5134 | |
| Silica | Sigma-Aldrich | S5631 | |
| Triton X 100 | BDH Chemicals | 14530 | |
| Diethylpyrocarbonate | Sigma-Aldrich | D-5758 | |
| QIAamp viral RNA Mini Kit (250) | QIAGEN | 52906 | |
| QuantiTec Probe RT-PCR kit (200) | QIAGEN | 204443 | |
| Qiagen One Step RT-PCR Kit (200) | QIAGEN | 210212 | |
| Rnase Inhibitor 2000 units | A.Biosystems | N808-0119 | 2000 unids/vial |
| Non-Stick Rnae-free Microfuge Tubes | Ambion | AM12450 | |
| UltraPure Agarose 1000 | Invitrogen | 16550-100 |
References
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