डीएनए निष्कर्षण के लिए एक सरल Chelex से प्रोटोकॉल<em> एनोफ़ेलीज़</em> एसपीपी.
Summary
एक तेजी से और सस्ती मच्छर नमूनों से गुणवत्ता मलेरिया परजीवी और वेक्टर डीएनए निकालने में वर्णित है. Chelating Chelex राल के गुण पर Capitalizing, सरल विधि मच्छर मध्य पेट और लार ग्रंथि चरणों में मलेरिया परजीवी के जीनोटाइपिंग, के रूप में के रूप में अच्छी तरह से की आणविक पहचान के लिए सक्षम बनाता है<em> एनोफ़ेलीज़</em> पीसीआर द्वारा प्रजातियों भाई.
Abstract
Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 μl of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 μl was retained while the other 10 μl was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min’ processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.
Protocol
Representative Results
Discussion
सरलीकृत Chelex यहाँ प्रस्तुत विधि गुणवत्ता एनोफ़ेलीज़ एसपीपी और पी. की निकासी के लिए सक्षम बनाता है फाल्सीपेरम मच्छर विविध पीसीआर अनुप्रयोगों के लिए उत्तरदायी नमूनों से डीएनए. इस तकनीक मलेरिया वेक्टर मच्छरों की आणविक पहचान और दवा प्रतिरोधी पी. की निगरानी के लिए नियोजित किया जा सकता है राष्ट्रीय मलेरिया नियंत्रण कार्यक्रम के लिए मच्छरों फाल्सीपेरम alleles. सरलीकृत Chelex तकनीक का लाभ सरलता, कम अभिकर्मकों और इसलिए लागत, सुरक्षा और कम नमक भुरकना विधि 10 (2 घंटा 47 मिनट और एक रात में कदम, टेबल 1) के रूप में इस तरह के मानक प्रोटोकॉल से प्रसंस्करण समय (37 मिनट) शामिल हैं. aforementioned फायदे और न्यूनतम अभिकर्मक आवश्यकताओं (3 अभिकर्मकों) वर्तमान मानक प्रोटोकॉल (10 अभिकर्मकों, 1 टेबल) 11,15 तुलना, सरलीकृत Chelex प्रोटोकॉल विशेष रूप से स्थानिकमारी वाले देश में, जहां एक निरंतर अभिकर्मक प्रयोगशालाओं के लिए अनुकूल बनाने केआपूर्ति श्रृंखला अक्सर को बनाए रखना मुश्किल है. विधि की एक सीमा है कि मानक प्रोटोकॉल की तरह, यह भी पीसीआर मच्छर 16 झिल्ली में हो जाता inhibitors के अधीन था. हालांकि, यह आसानी से BSA के assays में शामिल किए जाने से राहत मिली है. BSA भी सफलतापूर्वक किया गया है अन्य पीसीआर 17,18 अनुप्रयोगों में inhibitors के खिलाफ एक प्रवर्धन बढ़ाने के रूप में कार्यरत हैं.
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
लेखकों Macha के प्रमुखों headmen, और उनके unwaveing मच्छर क्षेत्र नमूना संग्रह के दौरान सहयोग के लिए समुदायों के लिए आभारी हैं. डॉ. डौग Norris और रिबका केंट बाहर नमक भुरकना प्रोटोकॉल पर अमूल्य विशेषज्ञता की पेशकश की. इस काम जॉन्स हॉपकिंस मलेरिया अनुसंधान संस्थान पायलट अनुदान प्रणाली द्वारा वित्त पोषित किया गया था. मच्छर और परजीवी डीएनए प्रयोगशाला मानकों कृपया MR4, अमेरिकी प्रकार और संस्कृति संग्रह द्वारा दान किया गया.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| Chelex-100, 50-100 dry mesh | BioRad | #143-2832 | |
| Saponin | SIGMA | 142-7425 | |
| BSA | New England Biolabs | B9001S |
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