The development and evaluation of a single step multiplex PCR method for simultaneous detection of Brugia malayi and Wuchereria bancrofti (original) (raw)
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Combined detection of Brugia malayi and Wuchereria bancrofti using single PCR
Acta Tropica, 2005
A single step PCR method has been developed for the combined detection of the human filarial parasites, Brugia malayi and Wuchereria bancrofti. Parasites' DNA were isolated from filaria positive blood samples that were collected from endemic areas. The primers used were Hha1 and Ssp I, which amplified the DNA fragments of 322 bp and 188 bp specific to B. malayi and W. bancrofti, respectively. The sensitivity of the assay was tested with blood and mosquito samples having one W. bancrofti in a pool of 10 B. malayi. The assay was further evaluated on field collected blood and mosquito samples. Use of this assay as a diagnostic tool for the detection of filariasis being the most promising aspect of this study, offers scope for detection of both the parasites even at low levels of infection.
The Korean Journal of Parasitology, 2013
A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2ºC, 79.0±0.3ºC, 76.8± 0.1ºC, and 79.9± 0.1ºC, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.
A REAL-TIME PCR-BASED ASSAY FOR DETECTION OF WUCHERERIA BANCROFTI DNA IN BLOOD AND MOSQUITOES
We developed and evaluated real-time polymerase chain reaction (PCR) assays for detecting Wuchereria bancrofti DNA in human blood and in mosquitoes. An assay based on detection of the W. bancrofti "LDR" repeat DNA sequence was more sensitive than an assay for Wolbachia 16S rDNA. The LDR-based assay was sensitive for detecting microfilarial DNA on dried membrane filters or on filter paper. We also compared real-time PCR with conventional PCR (C-PCR) for detecting W. bancrofti DNA in mosquito samples collected in endemic areas in Egypt and Papua New Guinea. Although the two methods had comparable sensitivity for detecting filarial DNA in reference samples, real-time PCR was more sensitive than C-PCR in practice with field samples. Other advantages of real-time PCR include its high-throughput capacity and decreased risk of crosscontamination between test samples. We believe that real-time PCR has great potential as a tool for monitoring progress in large-scale filariasis elimination programs.
Detection of Brugia malayi in mosquitoes by the polymerase chain reaction
Journal of the American Mosquito Control Association, 1998
Accurate identification of filarial parasites in mosquitoes poses a major problem for the coordination of filariasis control programs. Traditional methods are tedious, and some are not specific enough to give satisfactory results. Amplification of specific gene sequences by primer-directed polymerase chain reaction (PCR) has been increasingly utilized as a diagnostic tool. However, current protocols for the extraction of parasite DNA from mosquito samples are tedious and could lead to failure of PCR amplification. We demonstrate that the use of Chelex is an efficient method for DNA extraction from mosquitoes and the parasite and that PCR amplification with primers specific for Brugia malayi yields a band of the expected size. The PCR products were transferred to a nylon membrane with Southern blotting, and a B. malayi-specific digoxigenin-labeled probe confirmed the sequence similarity of the PCR-amplified fragment and increased the sensitivity of the PCR assay. Use of this probe en...
Transactions of The Royal Society of Tropical Medicine and Hygiene, 2001
Focally endemic bancroftian filariasis is targeted for elimination in the Nile delta of Egypt. Improved methods are needed for identifying endemic villages to be included in the control programme and for monitoring its success. We have evaluated the performance of a polymerase chain reaction (PCR) assay in estimating Wuchereria bancroft infection in pools of Culex pipiens (l-25 females) from 2 adjacent villages with high (El Qolzom, 10.8%) and low (Kafr Shorafa, 2.1%) prevalence rates of human filariasis. This assay detects a repeated sequence in W bumrofiideoxyribonucleic acid (DNA). Mosquitoes resting within houses were captured by aspiration and pooled by house. Houses were classified as positive or negative for human filarial infection based on night blood examinations of residents. The assay detected parasite DNA in mosquitoes from 60% of 25 infected houses and 24% of 25 uninfected houses. PCR processing of mosquitoes caught within houses of unknown filariasis infection status (44 in El Qolzom, 37 in Kafr Shorafa) identified 31.8% and 8.1% of houses, respectively, as containing infected mosquitoes. These results support the validity of the PCR assay for evaluating filarial prevalence in different villages. C. pipiens collected outdoors in dry ice-baited traps and tested by PCR (266 in Qolzom, 82 in Kafr Shorafa) did not contain parasite DNA. Pools of female mosquitoes (296 in Qolzom, 240 in Kafr Shorafa) captured in oviposition traps were also negative. We concluded that the PCR based assay is a powerful epidemiological tool that can be used for evaluating W. bancrofri infection in villages in the Nile delta and for monitoring the application of control programmes in filariasis endemic areas.
Parasitology Research, 2012
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Molecular and Biochemical Parasitology, 2013
With the Global Program for the Elimination of Lymphatic Filariasis continuing to make strides towards disease eradication, many locations endemic for the causative parasites of lymphatic filariasis are realizing a substantial decrease in levels of infection and rates of disease transmission. However, with measures of disease continuing to decline, the need for time-saving and economical molecular diagnostic assays capable of detecting low levels of parasite presence is increasing. This need is greatest in locations co-endemic for both Wuchereria bancrofti and Brugia parasites because testing for both causative agents individually results in significant increases in labor and reagent costs. Here we describe a multiplex, TaqMan-based, real-time PCR assay capable of simultaneously detecting W. bancrofti and Brugia malayi DNA extracted from human bloodspots or vector mosquito pools. With comparable sensitivity to established singleplex assays, this assay provides significant cost and labor savings for disease monitoring efforts in co-endemic locations.
Proceedings of the 4th International Symposium on Health Research (ISHR 2019), 2020
Mansonia is one of the genera of mosquitoes that play a role in the transmission of filariasis in North Hulu Sungai Utara undertook mass treatment of entire endemic communities from 2006 to 2015 to eliminate the transmission of the disease. However, post validation surveillance activities are required to ensure the gains achieved are sustained. Entomological survey was carried out to assess the mosquito vectors of the disease and determined the presence of infection in these vectors, testing the hypothesis that transmission has already been interrupted in North Hulu Sungai District. The study was conducted in Pihaung Village which is an endemic filariasis village, which adult filarial mosquito vectors were collected by using double net-trapped human bait method with modification. Abundance, species dominance were analyzed and PCR-based to determine DNA filarial of suspected vector. Total of 942 mosquitoes from five genera Anopheles, Aedes, Armigeres, Culex and Mansonia were captured at both sites. Of which, Mansonia has the highest relative abundance 29.96%. In this study periode resulted Mansonia dives Man Hour Density and Man Biting Rate were 4.7 and 10.5 respectively. PCR examination were detected Brugia malayi DNA on Mansonia as suspected vector in Pihaung Village. This study conclude the existing of Brugia malayi DNA in this area highlighting potential risk for the re-emergence of LF transmission. This study recommends routine vector control and strengthen surveillance is required to continuously monitor the filarial suspected vector density.
PLoS neglected tropical diseases, 2018
Currently, molecular xenomonitoring efforts for lymphatic filariasis rely on PCR or real-time PCR-based detection of Brugia malayi, Brugia timori and Wuchereria bancrofti in mosquito vectors. Most commonly, extraction of DNA from mosquitoes is performed using silica column-based technologies. However, such extractions are both time consuming and costly, and the diagnostic testing which follows typically requires expensive thermal cyclers or real-time PCR instruments. These expenses present significant challenges for laboratories in many endemic areas. Accordingly, in such locations, there exists a need for inexpensive, equipment-minimizing diagnostic options that can be transported to the field and implemented in minimal resource settings. Here we present a novel diagnostic approach for molecular xenomonitoring of filarial parasites in mosquitoes that uses a rapid, NaOH-based DNA extraction methodology coupled with a portable, battery powered PCR platform and a test strip-based DNA ...