Простой протокол Chelex для выделения ДНК из<em> Anopheles</em> SPP.
Summary
Быстрый и доступный способ извлечения паразита малярии качества и векторной ДНК из образцов комаров описано. Опираясь на свойства хелатирующие смолы Chelex, простой метод позволяет генотипирования малярийных паразитов в комара средней кишки и слюнных желез фаз, а также молекулярная идентификация<em> Anopheles</em> Видов-двойников с помощью ПЦР.
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
Упрощенный метод Chelex представленные здесь позволяет добыче качества Anopheles SPP и P. тропической ДНК из образцов комаров поддаются разнообразных приложений ПЦР. Эта техника может быть использована для молекулярной идентификации комаров переносчиком малярии и наблюдение за лекарственной устойчивостью P. аллелей в тропической комаров для национальных программ борьбы с малярией. Преимущества упрощенной техникой Chelex включают простоту, меньше реагентов и, следовательно, стоимости, безопасности и короче время обработки (37 мин), чем стандартные протоколы, такие как метод высаливания 10 (2 ч 47 мин и в течение ночи шага, таблица 1). Указанные преимущества и минимальные требования реагента (3 реагентов) по сравнению с текущим стандартным протоколом (10 реагентов, таблица 1) 11,15, сделать упрощенные Chelex протокола особенно дружелюбны к эндемичным лабораториях страны, где постоянно реагентовцепочками поставок часто трудно поддерживать. Ограничение метода является то, что, как стандартный протокол, это было также подлежат ПЦР ингибиторов известно, происходят в организме комара покрова 16. Тем не менее, это легко освобождается от включения BSA в анализах. BSA также успешно использоваться в качестве усиления усилителя с ингибиторами в других приложениях ПЦР 17,18.
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Авторы выражают благодарность начальников, старост и общин Macha для их unwaveing сотрудничают во комаров коллекции образцов поле. Доктор Дуг Норрис и Ревекка Kent предлагается бесценный опыт на высаливания протокол. Эта работа финансировалась по борьбе с малярией Johns Hopkins научно-исследовательский институт экспериментальной системы грантов. Комар и паразитов ДНК-лаборатория стандартов, любезно подаренных MR4, американский тип и культуры Collection.
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 |
Riferimenti
- Bonnet, M., et al. Efficacy of antimalarial treatment in Guinea: in vivo study of two artemisinin combination therapies in Dabola and molecular markers of resistance to sulphadoxine-pyrimethamine in N’Zerekore. Malar. J. 6, 54 (2007).
- Nsimba, B., et al. Sulphadoxine/pyrimethamine versus amodiaquine for treating uncomplicated childhood malaria in Gabon: a randomized trial to guide national policy. Malar. J. 7, 31 (2008).
- Oyewole, I. O., et al. Behaviour and population dynamics of the major anopheline vectors in a malaria endemic area in southern Nigeria. J. Vector Borne Dis. 44, 56-64 (2007).
- Harris, I., et al. A large proportion of asymptomatic Plasmodium infections with low and sub-microscopic parasite densities in the low transmission setting of Temotu Province, Solomon Islands: challenges for malaria diagnostics in an elimination setting. Malar. J. . 9, 254 (2010).
- Ouedraogo, A. L., et al. Substantial contribution of submicroscopical Plasmodium falciparum gametocyte carriage to the infectious reservoir in an area of seasonal transmission. PLoS ONE. 4, e8410 (2009).
- Birx, D., de Souza, M., Nkengasong, J. N. Laboratory challenges in the scaling up of HIV, TB, and malaria programs: The interaction of health and laboratory systems, clinical research, and service delivery. Am. J. Clin. Pathol. 131, 849-851 (2009).
- Ishengoma, D. R., et al. Health laboratories in the Tanga region of Tanzania: the quality of diagnostic services for malaria and other communicable diseases. Ann. Trop. Med. Parasitol. 103, 441-453 (2009).
- Alonso, P. L., et al. A research agenda for malaria eradication: vector control. PLoS Med. 8, e1000401 (2011).
- Grimberg, J., et al. A simple and efficient non-organic procedure for the isolation of genomic DNA from blood. Nucleic Acids Res. 17, 8390 (1989).
- Scott, J. A., Brogdon, W. G., Collins, F. H. Indentification of single specimens of the Anopheles gambiae complex by polymerase chain reaction. American Journal of Tropical Medicine and Hygiene. 49, 520-529 (1993).
- Kent, R. J., Norris, D. E. Identification of mammalian blood meals in mosquitoes by a multiplexed polymerase chain reaction targeting cytochrome B. Am. J. Trop. Med. Hyg. 73, 336-342 (2005).
- Duraisingh, M. T., Curtis, J., Warhurst, D. C. Plasmodium falciparum: detection of polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes by PCR and restriction digestion. Exp. Parasitol. 89, 1-8 (1998).
- Gorrochotegui-Escalante, N., Munoz, M. L., Fernandez-Salas, I., Beaty, B. J., Black, W. C. T. Genetic isolation by distance among Aedes aegypti populations along the northeastern coast of Mexico. Am. J. Trop. Med. Hyg. 62, 200-209 (2000).
- Kreader, C. A. Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl. Environ. Microbiol. 62, 1102-1106 (1996).
- Norris, D. E., Shurtleff, A. C., Toure, Y. T., Lanzaro, G. C. Microsatellite DNA polymorphism and heterozygosity among field and laboratory populations of Anopheles gambiae ss (Diptera Culicidae). J. Med. Entomol. 38, 336-340 (2001).
- Schriefer, M. E., Sacci, J. B., Wirtz, R. A., Azad, A. F. Detection of polymerase chain reaction-amplified malarial DNA in infected blood and individual mosquitoes. Exp. Parasitol. 73 (91), 311-316 (1991).
- Al-Soud, W. A., Radstrom, P. Purification and characterization of PCR-inhibitory components in blood cells. J. Clin. Microbiol. 39, 485-493 (2001).
- Hyun, C., Filippich, L. J., Hughes, I. The inhibitory effect of pentobarbitone on reverse transcription-PCR. J. Biochem. Biophys. Methods. 62, 63-68 (2005).
