Abstract
There is a need for an endogenous internal control (EIC) for PCRs to monitor the quality and quantity of DNA in test samples. We designed and validated a fluorescence resonance energy transfer (FRET)-PCR targeting the mammalian homolog of the hydroxymethylbilane synthase (HMBS) gene as an EIC for PCRs on mammals. The designed FRET-PCR detected the HMBS gene in whole blood of 13 mammalian species collected from eight countries and in 11 murine organs/tissues. It could also be used to quantify the volumes of mammalian blood meals in mosquitoes and by sequencing the amplicons obtained we could determine the mammalian species (6) from which the meal was obtained. The FRET-PCR proved highly sensitive (one gene copy in 0.05 ng tissue or 0.5 nl whole blood) and specific with no false negative or positive results. The high sensitivity and specificity of the FRET-PCR and its ability to differentiate mammalian species makes it an ideal EIC for PCRs involving mammals and a useful tool for hematophagous insect studies.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Atmadja DS, Tatsuno Y, Ueno Y, Nishimura A (1995) The effect of extraction methods. The kind of organ samples and the examination delay on the DNA yields and typing. Kobe J Med Sci 41:197–211
Bruggemann J, Stephen J, Chang YJ, Macnaughton S, Kowalchuk G, Kline E, White DC (2000) Competitive PCR-DGGE analysis of bacterial mixtures an internal standard and an appraisal of template enumeration accuracy. J Microbiol Methods 40:111–123
Burggraf S, Olgemöller B (2004) Simple technique for internal control of real-time amplification assays. Clin Chem 50:819–825
Caboux E, Lallemand C, Ferro G, He´mon B, Mendy M, Biessy C, Sims M, Wareham N, Britten A, Boland A, Hutchinson A, Siddiq A, Vineis P, Riboli E, Romieu I, Rinaldi S, Gunter MJ, Peeters PH, van der Schouw YT, Travis R, Bueno-de-Mesquita HB, Canzian F, Sánchez MJ, Skeie G, Olsen KS, Lund E, Bilbao R, Sala N, Barricarte A, Palli D, Navarro C, Panico S, Redondo ML, Polidoro S, Dossus L, Boutron-Ruault MC, Clavel-Chapelon F, Trichopoulou A, Trichopoulos D, Lagiou P, Boeing H, Fisher E, Tumino R, Agnoli C, Hainaut P (2012) Sources of pre-analytical variations in yield of DNA extracted from blood samples: analysis of 50,000 DNA samples in EPIC. PLoS One 7:e39821
CDC (2013) Pictorial keys to arthropods, reptiles, birds, mammals of public health significance. www.cdc.gov/nceh/ehs/Publications/Pictorial_Keys.htm. Assessed 10 January 2014
Cui X, Zhou J, Qiu J, Johnson MR, Mruga M (2009) Validation of endogenous internal real-time PCR controls in renal tissues. Am J Nephrol 30:413–417
Huggett J, Dheda K, Bustin S, Zumla A (2005) Real-time RT-PCR normalisation: strategies and considerations. Genes Immunol 6:279–284
Kalle E, Gulevich A, Rensing C (2013) External and semi-internal controls for PCR amplification of homologous sequences in mixed templates. J Microbiol Methods 95:285–294
Kaltenboeck B, Wang C (2005) Advances in real-time PCR: application to clinical laboratory diagnostics. Adv Clin Chem 40:219–259
Kelly PJ, Xu C, Lucas H, Loftis A, Abete J, Zeoli F, Stevens A, Jaegersen K, Ackerson K, Gessner A, Kaltenboeck B, Wang C (2013) Ehrlichiosis, babesiosis, anaplasmosis and hepatozoonosis in dogs from St. Kitts, West Indies. PLoS One 8:e53450
Petersen D, Dahllöf I (2005) Improvements for comparative analysis of changes in diversity of microbial communities using internal standards in PCR-DGGE. FEMS Microbiol Ecol 53:339–348
Rosenstraus M, Wang Z, Chang SY, DeBonville D, Spadoro JP (1998) An internal control for routine diagnostic PCR: design, properties, and effect on clinical performance. J Clin Microbiol 36:191–197
Wang C, Gao D, Vaglenov A, Kaltenboeck B (2004) One-step real-time duplex reverse transcription PCRs simultaneously quantify analyte and housekeeping gene mRNAs. Biotechniques 36(508–516):518–519
Wang C, Mount J, Butler J, Gao D, Jung E, Blagburn BL, Kaltenboeck B (2012) Real-time PCR of the mammalian hydroxymethylbilane synthase (HMBS) gene for analysis of flea (Ctenocephalides felis) feeding patterns on dogs. Parasitol Vectors 5:4
Zhang J, Wei L, Kelly P, Freeman M, Jaegerson K, Gong J, Xu B, Pan Z, Xu C, Wang C (2013) Detection of Salmonella spp. using a generic and differential FRET-PCR. PLoS One 8:e76053
Zhu G, Xia H, Zhou H, Li J, Lu F, Liu Y, Cao J, Gao Q, Sattabongkot J (2013) Susceptibility of Anopheles sinensis to Plasmodium vivax in malarial outbreak areas of central China. Parasitol Vectors 6:176
Acknowledgments
This project was supported by a grant from the National Natural Science Foundation of China (No. 31272575) and by the Ross University School of Veterinary Medicine.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Wei, L., Kelly, P., Zhang, J. et al. Use of a universal hydroxymethylbilane synthase (HMBS)-based PCR as an endogenous internal control and to enable typing of mammalian DNAs. Appl Microbiol Biotechnol 98, 5579–5587 (2014). https://doi.org/10.1007/s00253-014-5659-x
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-014-5659-x