Use Of A Universal Hydroxymethylbilane Synthase (HMBS) Based FRET-qPCR As An Endogenous Internal Control For Mammalian DNAS And Its Application

As a specific and sensitive molecular detection tool, the polymerase chain reaction (PCR) has been widely used on biological research areas worldwide. However, it is well known that many factors can adversely influence the quantity and quality of DNA and result in false positivity or negativity during the whole process of sample collection, transportation, conservation, DNA extraction and PCR detection. Therefore, there is an urgent need to develelop an endogenous internal control (EIC) system for dynamic monitoring of the quality and quantity of DNAs for PCRs.In study one of this thesis, we designed and validated a fluorescence resonance energy transfer (FRET) quantitative PCR (qPCR) targeting the mammalian homolog of the hydroxymethylbilane synthase (HMBS) gene as an EIC for PCRs on mammals. In studies two and three, EIC was applied to monitor the quality and quantity of tested DNAs during the molecular epidemiological investigation of seven vector-borne diseases in dogs from Nicaragua and Costa Rica.The designed EIC could detect whole blood of human and other 12 mammalian species (dog, cat, cattle, mouse, pig, donkey, horse, monkey, sheep, goat, lion and cheetah) collected from 8 countries and the HMBS gene in 11 murine organs/tissues (heart, liver, brain, spleen, lung, adipose, kidney, muscle, hair, hone and skin). It was also used conveniently on mosquitoes to determine the volumes of mammalian blood meals, and determine the mammalian species from which whole blood was obtained. Sequencing of amplicons obtained from mosquitoes in this investigation revealed they had ingested blood from six mammals. The FRET-qPCR proved highly sensitive (one gene copy in 0.05 ng tissue or 0.5 nl whole blood/reaction) and specific with no false negative or positive results. The high sensitivity and specificity of the FRET-qPCR and its ability to differentiate mammalian species makes it an ideal EIC for PCRs involving mammals and a useful tool for hematophagous insect studies.Though vector-borne diseases are important causes of morbidity and mortality in dogs in tropical areas, there is little information on these conditions in Central America. Several FRET-qPCRs for seven vector-borne pathogens were performed on a Roche LightCycler PCR Instrument to investigate their prevalence in convenience samples of whole blood from apparently healthy dogs in Nicaragua and Costa Rica.In Nicaragua, we found DNAs of Rickettsia (R.) felis (5%), Babesia (B.) spp. (26%), Hepatozoon (H.) canis (51%), Anaplasma (A.) platys (13%) and Ehrlichia (E.) canis (56%) in the 39 dogs studied. The qPCRs for Coxiella (C.) burnetii and Dirofilaria (D.) immitis were negative. Of the 30 (80%) dogs that were positive by qPCR,12 (31%) were positive for one agent,11 (28%) for two,3 (8%) for three, and 4 (10%) for four agents.In PCRs testing on blood from dogs in Costa Rica we did not detect DNAs of R. felis and C. burnetii but we did find evidence of infection with D. immitis (22.5%), H. canis (37.5%), B. spp. (25%), A. platys (7.5%) and E. canis (50%). Nine dogs (22.5%) were free of any vector-borne pathogens while 14 (35%) were infected with a single pathogen,11 (27.5%) with two,4 (10%) with three, and 1 (2.5%) with five pathogens.In conclusion, we successfully established a HMBS-based PCR as an EIC, enabling the monitor of the whole process of PCRs. With high sensitivity and specificity, the system can be widely applied on a variety of mammalian samples. When serving as the EIC in the molecular epidemiological survey of vector-borne diseases, it helped to guarantee that the DNAs were appropriate for PCRs. As the results show, dogs in Nicaragua and Costa Rica are commonly infected with a variety of vector-borne pathogens, some of which may also infect people. This is also the first report of B. gibsoni in dogs from Central America and the first record of vector-borne agents in dogs from Nicaragua.