INTRODUCTION
Lymphatic filariasis, a mosquito-borne disease, is a major public health problem, particularly in the tropics and subtropics. It is caused by the nematodes Wuchereria bancrofti, Brugia malayi, and Brugia timori.1,2 Symptoms include acute fever and chill and chronic lymphoedema and hydroceles. Brugian filariasis caused by B. malayi and B. timori affects ~13 million people, mainly in India and Southeast Asia.3 A principal goal of the Global Program to Eliminate Lymphatic Filariasis (GPELF) is interruption of the transmission of infection.2 Hence, the availability of efficient and effective diagnostic tools to monitor the presence or absence of filarial larvae in the mosquito vector is particularly important.4 Entomologic methods for the detection of filarial larvae in mosquito vectors are based on the dissection of mosquitoes. However, these methods are laborious, tedious, and time consuming and carry a low sensitivity and a need for specially trained microscopists.
Many conventional polymerase chain reaction (c-PCR) assays have been developed to detect filarial DNA in human blood and mosquito vectors.4–6 All of these procedures require agarose gel electrophoresis for the analysis. However, determination by gel electrophoresis is slow, has a limited throughput, and is prone to carry-over contamination and subjective results. Recently, effective real-time PCR has greatly improved molecular detection and differential diagnosis of microorganisms belonging to the same genus and has increasingly replaced c-PCR. Effective real-time PCR is not only accurate, sensitive, fast, and can quantify specific DNA in biologic samples,7 but it also differentiates species or strains of several medically pathogenic microorganisms by melting curve analysis.8–10 Moreover, this method provides a high-throughput means because it does not need agarose gel electrophoresis for analysis of the amplicons and has a broad dynamic range.11 The method has great potential for epidemiologic studies and for monitoring elimination programs of infectious agents. Recently, W. bancrofti and Brugia spp. DNA have been shown in infected blood and in infected mosquito vectors by either a Taqman probe or an Eclipse minor groove binding probe based on real-time PCR.3,12,13
In contrast to the reports by Rao and others3,12 and Fischer and others,13 another assay format using real-time fluorescence resonance energy transfer (FRET) PCR combined with a melting curve analysis has been reported by our group for W. bancrofti DNA detection in mosquito vectors.14 This format can be used for a differential detection of W. bancrofti DNA from DNA of Dirofilaria immitis and B. malayi and from human and mosquito vectors. However, B. malayi DNA detection based on this technique is still not feasible. Here, we have used specific primers amplifying the repetitive sequence, HhaI repeat, of B. malayi (GenBank accession no. M12691)15 and developed a genus specific and sensitive real-time FRET PCR using a LightCycler-based PCR system (Roche Applied Science, Mannheim, Germany) for the differential detection of larval stages of B. malayi in mosquitoes. Two individually fluorophore-labeled specific hybridization probes were used, and melting point profiles were compared with the effects gained from other control DNA.
MATERIALS AND METHODS
Mosquitoes.
Aedes togoi mosquitoes, a mosquito species from Koh Nom Sao, Chanthaburi Province, Eastern Thailand,16 were artificially infected with B. malayi. The mosquito larvae were taken from their breeding places and reared in the insectarium.
Blood sources for mosquitoes.
Blood infected with microfilariae of nocturnally subperiodic B. malayi was obtained from an infected cat. The worms were originally taken from a 20-year-old woman in Bang Paw District, Narathiwat Province, Southern Thailand, and used to experimentally infect domestic cats and are now kept in the Department of Parasitology, Faculty of Medicine, Chiang Mai University. The maintenance and care of animal experiments in this study complies with the current Thai laws.
Infection of mosquitoes.
The B. malayi–infected mosquitoes were obtained as previously described.17 Briefly, blood was drawn from the femoral vein of the infected cat, and heparin was added at a concentration of 10 units/mL blood. Next, 3- to 5-day-old female Ae. togoi mosquitoes (fasted for 12 hr) were allowed to feed on the heparinized blood using an artificial membrane feeding technique as described previously.18 To ensure infectivity, the mosquitoes were dissected in normal saline solution 14 days after feeding, and the number of larvae was counted under a dissecting microscope. Only infected mosquitoes were used for the experiments, and the number of parasites was recorded. After dissection, all B. malayi larvae and the body of the individual infected mosquito were mixed and placed in a 1.5-mL microcentrifuge tube, labeled, and kept at −20°C for DNA extraction. The range and mean ± SD of the number of parasites per infected mosquito were 1–42 and 7.06 ± 10.35 larvae, respectively.
Source of DNA for specificity evaluation.
Adult worms of D. immitis from infected dogs (from Khon Kaen Province), uninfected Ae. togoi, uninfected Culex pipiens quinquefasciatus, and Plasmodium falciparum–infected human red blood cells and human leukocytes were separately extracted and purified using the Nucleospin Tissue kit (Macherey-Nagel, Duren, Germany) according to the manufacturer’s recommendations. The DNA was resuspended in 100 μL of 5 mmol/L Tris-HCl, pH 8.5, and used for specificity evaluation. The Cx. quinquefasciatus mosquitoes infected with the nocturnally periodic W. bancrofti (from a Burmese immigrant, Tak Province, northwestern Thailand), obtained as previously described,14 were also used for DNA extraction as described above. The range and mean ± SD of the parasite load per infected mosquito were 1–18 and 5.36 ± 5.16 parasites, respectively.
Preparation of specimens for real-time FRET PCR.
Each specimen, infected or uninfected, was put in a 1.5-mL micro-centrifuge tube and homogenized with disposable polypropylene pestles (Bellco Glass, Vineland, NJ), followed by extraction using the Nucleospin Tissue kit (Macherey-Nagel). The DNA was eluted in 100 μL of 5 mmol/L Tris-HCl, pH 8.5, of which 5 μL were used in the real-time PCR reaction.
Real-time FRET PCR assay.
The LightCycler PCR and detection system (Roche Applied Science, Mannheim, Germany) was used for amplification and quantification. PCR was performed in glass capillaries. A specific primer pair, BM-F (5′-TCATTAGACAAGGATATTGGTTC-3′) and BM-R (5′-TTTAAACTATAAAATGACAACACA-3′) (Tib Molbiol, Berlin, Germany), was designed to bind to the HhaI repetitive sequence of the B. malayi genome as described previously (GenBank accession no. M12691).15 The reasons of using this target sequence were 1) the sequence was organized as a tandem repeat and presented at several thousand copies per haploid genome19 and 2) several c-PCR tests for the detection of Brugia DNA have used this sequence as a target, and no sequence variation has been shown in different strains of B. malayi and B. timori.20,21
For amplification detection, the LightCycler FastStart DNA Master HybProbe Kit was used as recommended by the manufacturer. Briefly, a pair of adjacent oligoprobes was hybridized to an internal genus-specific repetitive sequence of B. malayi. One probe was labeled at the 5′ end with the LightCycler Red 705 fluorophore (5′-Red 705-TGTACCAGTGCTGGTCGTGTA-Phosphate-3′; BMLC705 probe), and the other was labeled at the 3′ end with 530 fluorescein (5′-AAATTAATTGACTATGTTACGTGAA-Flou 530-3′; BMFL530 probe; Tib Molbiol). Probes and primers were designed by using the LC probe design software (Roche Applied Science). Schematic diagram of the hybridization analyzes used in the assay is shown in Figure 1. When the probes hybridized to the same DNA strand internal to the PCR primers, the probes came in close proximity and produced a fluorescence resonance energy transfer (FRET). During FRET, the 530 fluorescein was excited by the light source of the LightCycler instrument. The excitation energy was transferred to the acceptor fluorophore of the LightCycler Red 705 only when it is positioned in close vicinity of the former and the emitted fluorescence was measured after annealing by the photohybrids of the instrument. After a complete PCR running, it is possible to perform a melting point analysis, during which the temperature is lowered below the annealing temperature of the probes and slowly increased. The fluorescence signal decreases when the probe melts off its target.
The PCR mixture contained LightCycler FastStart DNA Master HybProbe (Roche Applied Science), 5 mmol/L MgCl2, 0.5 μmol/L BM-F primer, 0.5 μmol/L BM-R primer, 0.4 μmol/L BMLC705 probe, and 0.2 μmol/L BMFL530 probe, respectively. The total reaction volume was 20 μL. Samples were run by performing 45 cycles of repeated denaturation (5 seconds at 95°C), annealing (15 seconds at 45°C), and extension (8 seconds at 72°C). The temperature transition rate was 20°C/s. After amplification, a melting curve was produced by heating the product at 20°C/s to 95°C, cooling it to 40°C, keeping it at 40°C for 30 seconds, and heating it slowly at 0.1°C/s to 85°C. The fluorescence change intensity was measured throughout the slow heating phase. To determine the specificity of the oligonucleotide hybridization based on the FRET technique, DNA extracted from D. immitis, human leukocytes, uninfected Ae. togoi and Cx. quinquefasciatus, P. falciparum–infected human red blood cells, and W. bancrofti–infected Cx. quinquefasciatus were separately analyzed. Each run contained at least one negative control consisting of 5 μL distilled water.
For improved visualization of the melting temperatures (Tm), melting peaks were derived as previously described.14 Melting curves were used to determine the specific PCR products, which were confirmed by conventional agarose gel electrophoresis. The target sequence copy number represented by the cycle number was explained as the number of PCR cycles required for the fluorescence signal of the amplicons to exceed the detection threshold value. The correlation between worm loads and cycle numbers was analyzed by the Spearman rank correlation test.
Brugia malayi–positive control plasmid.
A positive control plasmid was constructed by cloning a PCR product of the B. malayi HhaI repeat15 into the pCR4-TOPO vector (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. The PCR products were obtained by c-PCR using primers BM-F (5′-TCATTAGACAAGGATATTGGTTC-3′) and BM-R (5′-TTTAAACTATAAAATGACAACACA-3′) (Tib Molbiol). The plasmid was propagated in Escherichia coli, and the nucleotide sequence of the inserted gene was sequenced in both directions. The nucleotide sequence of the cloned HhaI repeat revealed an identical structure to the B. malayi genome (GenBank accession no. M12691). The size of the plasmid was 4,109 bp (including the 153-bp HhaI sequence).
RESULTS
Standardization of the real-time FRET PCR.
Five microliters of serial dilutions (4 to 4 × 1010 copies) of B. malayi–positive control plasmid in water was tested to assess the sensitivity of hybridization real-time PCR. When using cycle numbers of 35 as the cut-off limit of detection, the reliable detection limit of the HhaI repeat target DNA sequence was ~4 × 102 copies of positive control plasmid (Figure 2). No fluorescence signal was obtained when purified DNA from W. bancrofti–infected Cx. quinquefasciatus, D. immitis, uninfected Ae. togoi and Cx. quinquefasciatus, P. falciparum–infected human red blood cells, or human leukocytes were tested.
To determine the capability of detection by real-time FRET PCR, each pool of 10, 30, 60, and 100 uninfected Ae. togoi adult mosquitoes inoculated with one infected mosquito that harbored one B. malayi larva were used. These samples were used for DNA extraction and examined the real-time FRET PCR assay. This method could detect filarial DNA of as little as one B. malayi larva infected in one mosquito inoculated in 100 uninfected Ae. togoi (data not shown).
Real-time FRET PCR to detect B. malayi in mosquitoes.
We used the Brugia-specific DNA sequence HhaI repeat to detect B. malayi in infected mosquitoes using the real-time FRET PCR assay combined with a melting curve analysis of the PCR product. A total of 30 B. malayi–infected Ae. togoi, 30 uninfected Ae. togoi, and 30 W. bancrofti–infected Cx. quinquefasciatus were separately determined. The real-time PCR could detect as little as one larva in a single mosquito. The melting curve analysis is shown in Figure 3. When using B. malayi–specific primers and probes, the mean ± SD of Tm values of B. malayi was 57.31 ± 0.07 (N = 30), whereas neither Tm value was shown in W. bancroft–infected and uninfected mosquito groups or other control DNAs, respectively. A total of 30 B. malayi–infected Ae. togoi were positive by melting curve analysis, whereas all 30 uninfected Ae. togoi and 30 W. bancrofti–infected C. quinquefasciatus were negative by melting curve analysis. The sensitivity and specificity were both 100%.
All B. malayi–infected mosquitoes and positive control plasmids were amplified by real-time PCR (Figure 4, Lanes 2–7) and showed the prominent 153-bp product, whereas genomic DNA from uninfected Ae. togoi (Figure 4, Lane 8), W. bancrofti–infected (Figure 4, Lanes 9–12) and uninfected (Figure 4, Lane 13), Cx. quniquefasciatus, and P. falciparum–infected human red blood cells (Figure 4, Lane 15) and human leukocytes (Figure 4, Lane 16) were amplified the faint bands. An ~300-bp amplification product was seen with D. immitis DNA (Figure 4, Lane 14). A significant correlation between cycle number and parasite load in mosquito controls is shown in Figure 5 (R = −0.582; P < 0.01). These results showed that real-time PCR can provide data on parasite densities in vectors.
DISCUSSION
Molecular xenomonitoring in mosquito vectors is another diagnostic choice for the GPELF.2 It can be used for detecting lymphatic filariasis-endemic areas and selecting regions for inclusion in the program. Although the classic method for the detection of filarial parasites in mosquitoes, dissection, is an inexpensive method, it requires technicians well trained in the identification of larvae in dissected mosquitoes. This classic method of dissection becomes increasingly inefficient as the prevalence of infection in the vector population decreases,4 as well as impractical for regions with low vector infection rates after mass drug administrations.2 c-PCR has previously been suggested as a promising tool for monitoring the work progress of eliminating lymphatic filariasis.4 Real-time PCR provides additional value to these procedures because it is highly specific and sensitive, allows a high throughput, and can be done on very small samples.12–14 In this study, we developed a real-time FRET PCR combined with a melting curve analysis to detect B. malayi DNA in infected mosquitoes. Two primers were used to produce the genus-specific amplicon, which was subsequently shown by its combined melting peak profile with two hybridization fluorophore-labeled probes. The method can detect as little as one filarial larva harbored in one infected mosquito inoculated in one pool of 100 uninfected mosquitoes. The result showed high sensitivity of method and possibly can be useful to apply for detection in field samples. In addition, this report described the first use of real-time FRET PCR for detection of B. malayi in mosquito vectors.
This assay differentiated B. malayi DNA in infected vectors from DNA of D. immitis and W. bancrofti. However, faint bands were shown with other control DNA (Figure 4, Lanes 8–13, 15, and 16) and an ~300-bp amplification product was seen with D. immitis DNA (Figure 4, Lane 14). This does not cause a problem because no specific fluorescence signal was shown during melting peak analysis. The above described technique showed a 100% sensitivity and specificity, and merging it with a melting curve analysis offered a rapid and reliable procedure for differentially identifying lymphatic filariasis. Moreover, the real-time FRET PCR used for amplifying the repetitive sequence, HhaI repeat, of B. malayi (Gen-Bank accession no. M12691) showed a correlation between the cycle numbers and the parasite numbers in the mosquitoes (Figure 5). This information suggests a quantitative result; however, results should be interpreted carefully because PCR can not differentiate between parasite stages, and the amount of DNA may vary between stages. Nevertheless, our assay represents another useful choice for the detection of Brugia DNA.
Recently, Fischer and others13 showed the persistence of B. malayi DNA in vector and non-vector mosquitoes by Taqman probe or an Eclipse minor groove binding probe based on real-time PCR. This does not pose a problem if xenomonitoring is done selectively in vectors only. For epidemiologic studies and eradication programs, confirmation assays detecting Brugia DNA in the blood of the population at risk and transmission monitoring of vectors need to be done. Although Brugia DNA detection is only an indirect measure of transmission, it does provide information on rates of lymphatic filarial transmission or the potential of transmission.
In summary, this study shows that real-time FRET PCR and melting curve analysis are sensitive and specific methods for Brugia DNA detection. The assay can be used to screen mosquito vectors for mapping areas of endemicity or human blood specimens for diagnosis. The method should be helpful for evaluating changes in transmission after mass drug administration and should be an important diagnostic tool for the GPELF.
Schematic illustration of PCR primers (BM-F and BM-R primers), anchor, and detection probes for the HhaI repetitive sequence of Brugia malayi. The probe BMFL530 is labeled with 530 fluorescein at the 3′ end and serves as anchor probe for the sensor BMLC705 probe. The sensor probe is labeled with LightCycler Red 705 fluorophore (LC red 705) at the 5′ end. Circle, 530 fluorescein; double circle, LC red 705.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 3; 10.4269/ajtmh.2008.78.509
Schematic illustration of PCR primers (BM-F and BM-R primers), anchor, and detection probes for the HhaI repetitive sequence of Brugia malayi. The probe BMFL530 is labeled with 530 fluorescein at the 3′ end and serves as anchor probe for the sensor BMLC705 probe. The sensor probe is labeled with LightCycler Red 705 fluorophore (LC red 705) at the 5′ end. Circle, 530 fluorescein; double circle, LC red 705.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 3; 10.4269/ajtmh.2008.78.509
Schematic illustration of PCR primers (BM-F and BM-R primers), anchor, and detection probes for the HhaI repetitive sequence of Brugia malayi. The probe BMFL530 is labeled with 530 fluorescein at the 3′ end and serves as anchor probe for the sensor BMLC705 probe. The sensor probe is labeled with LightCycler Red 705 fluorophore (LC red 705) at the 5′ end. Circle, 530 fluorescein; double circle, LC red 705.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 3; 10.4269/ajtmh.2008.78.509
Amplification plots of fluorescence (y-axis) vs cycle numbers (x-axis) show the analytical sensitivity of real-time PCR for detecting B. malayi plasmid DNA. A, B. malayi plasmid 4 × 1010 copies/reaction. B, B. malayi plasmid 4 × 108 copies/reaction. C, B. malayi plasmid 4 × 106 copies/reaction. D, B. malayi plasmid 4 × 104 copies/reaction. E, B. malayi plasmid 4 × 102 copies/reaction. F, B. malayi plasmid 4 × 10 copies/reaction. G, B. malayi plasmid 4 copies/reaction. H, Distilled water.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 3; 10.4269/ajtmh.2008.78.509
Amplification plots of fluorescence (y-axis) vs cycle numbers (x-axis) show the analytical sensitivity of real-time PCR for detecting B. malayi plasmid DNA. A, B. malayi plasmid 4 × 1010 copies/reaction. B, B. malayi plasmid 4 × 108 copies/reaction. C, B. malayi plasmid 4 × 106 copies/reaction. D, B. malayi plasmid 4 × 104 copies/reaction. E, B. malayi plasmid 4 × 102 copies/reaction. F, B. malayi plasmid 4 × 10 copies/reaction. G, B. malayi plasmid 4 copies/reaction. H, Distilled water.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 3; 10.4269/ajtmh.2008.78.509
Amplification plots of fluorescence (y-axis) vs cycle numbers (x-axis) show the analytical sensitivity of real-time PCR for detecting B. malayi plasmid DNA. A, B. malayi plasmid 4 × 1010 copies/reaction. B, B. malayi plasmid 4 × 108 copies/reaction. C, B. malayi plasmid 4 × 106 copies/reaction. D, B. malayi plasmid 4 × 104 copies/reaction. E, B. malayi plasmid 4 × 102 copies/reaction. F, B. malayi plasmid 4 × 10 copies/reaction. G, B. malayi plasmid 4 copies/reaction. H, Distilled water.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 3; 10.4269/ajtmh.2008.78.509
Representative melting curve analysis of two fluorophore-labeled probes hybridized to the amplification products of HhaI repeat DNA from B. malayi. The melting temperature (Tm) of the double-stranded fragment is visualized by plotting the negative derivative of the change in fluorescence divided by the change in temperature in relation to the temperature [− (d/dT) fluorescence (705/530)]. The turning point of this converted melting curve results in a peak and permits easy identification of the fragment-specific Tm. A, melting peaks of positive control plasmid, B, B. malayi–infected mosquitoes, and C, genomic DNA from D. immitis, W. bancrofti–infected Cx. pipiens quinquefasciatus and uninfected Ae. togoi, P. falciparum–infected human red blood cells, and human leukocytes, as well as the negative control containing no DNA.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 3; 10.4269/ajtmh.2008.78.509
Representative melting curve analysis of two fluorophore-labeled probes hybridized to the amplification products of HhaI repeat DNA from B. malayi. The melting temperature (Tm) of the double-stranded fragment is visualized by plotting the negative derivative of the change in fluorescence divided by the change in temperature in relation to the temperature [− (d/dT) fluorescence (705/530)]. The turning point of this converted melting curve results in a peak and permits easy identification of the fragment-specific Tm. A, melting peaks of positive control plasmid, B, B. malayi–infected mosquitoes, and C, genomic DNA from D. immitis, W. bancrofti–infected Cx. pipiens quinquefasciatus and uninfected Ae. togoi, P. falciparum–infected human red blood cells, and human leukocytes, as well as the negative control containing no DNA.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 3; 10.4269/ajtmh.2008.78.509
Representative melting curve analysis of two fluorophore-labeled probes hybridized to the amplification products of HhaI repeat DNA from B. malayi. The melting temperature (Tm) of the double-stranded fragment is visualized by plotting the negative derivative of the change in fluorescence divided by the change in temperature in relation to the temperature [− (d/dT) fluorescence (705/530)]. The turning point of this converted melting curve results in a peak and permits easy identification of the fragment-specific Tm. A, melting peaks of positive control plasmid, B, B. malayi–infected mosquitoes, and C, genomic DNA from D. immitis, W. bancrofti–infected Cx. pipiens quinquefasciatus and uninfected Ae. togoi, P. falciparum–infected human red blood cells, and human leukocytes, as well as the negative control containing no DNA.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 3; 10.4269/ajtmh.2008.78.509
Ethidium bromide staining patterns of the PCR products on a 1.5% agarose gel. Arrow indicates 153-bp B. malayi–specific band. Lane 1, negative control containing no DNA; Lane 2, PCR products obtained from positive control plasmid; Lanes 3–7, PCR products obtained from B. malayi–infected mosquitoes; Lane 8, PCR products from uninfected Ae. togoi; Lanes 9–12, PCR products from W. bancrofti–infected mosquitoes; Lane 13, PCR product from uninfected Cx. pipiens quinquefasciatus; Lane 14, PCR product from D. immitis; Lane 15, PCR product from P. falciparum–infected human red blood cells; Lane 16, PCR product from human leukocyte genomic DNA; Lane M, DNA size markers (1 kb plus DNA ladder from Invitrogen).
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 3; 10.4269/ajtmh.2008.78.509
Ethidium bromide staining patterns of the PCR products on a 1.5% agarose gel. Arrow indicates 153-bp B. malayi–specific band. Lane 1, negative control containing no DNA; Lane 2, PCR products obtained from positive control plasmid; Lanes 3–7, PCR products obtained from B. malayi–infected mosquitoes; Lane 8, PCR products from uninfected Ae. togoi; Lanes 9–12, PCR products from W. bancrofti–infected mosquitoes; Lane 13, PCR product from uninfected Cx. pipiens quinquefasciatus; Lane 14, PCR product from D. immitis; Lane 15, PCR product from P. falciparum–infected human red blood cells; Lane 16, PCR product from human leukocyte genomic DNA; Lane M, DNA size markers (1 kb plus DNA ladder from Invitrogen).
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 3; 10.4269/ajtmh.2008.78.509
Ethidium bromide staining patterns of the PCR products on a 1.5% agarose gel. Arrow indicates 153-bp B. malayi–specific band. Lane 1, negative control containing no DNA; Lane 2, PCR products obtained from positive control plasmid; Lanes 3–7, PCR products obtained from B. malayi–infected mosquitoes; Lane 8, PCR products from uninfected Ae. togoi; Lanes 9–12, PCR products from W. bancrofti–infected mosquitoes; Lane 13, PCR product from uninfected Cx. pipiens quinquefasciatus; Lane 14, PCR product from D. immitis; Lane 15, PCR product from P. falciparum–infected human red blood cells; Lane 16, PCR product from human leukocyte genomic DNA; Lane M, DNA size markers (1 kb plus DNA ladder from Invitrogen).
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 3; 10.4269/ajtmh.2008.78.509
The number of larvae (y-axis) plotted against the cycle numbers (x-axis). Negative correlations were observed between the number of larvae and cycle numbers (Spearman rank correlation test, R = −0.582; P < 0.01).
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 3; 10.4269/ajtmh.2008.78.509
The number of larvae (y-axis) plotted against the cycle numbers (x-axis). Negative correlations were observed between the number of larvae and cycle numbers (Spearman rank correlation test, R = −0.582; P < 0.01).
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 3; 10.4269/ajtmh.2008.78.509
The number of larvae (y-axis) plotted against the cycle numbers (x-axis). Negative correlations were observed between the number of larvae and cycle numbers (Spearman rank correlation test, R = −0.582; P < 0.01).
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 3; 10.4269/ajtmh.2008.78.509
Address correspondence to Wanchai Maleewong, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. E-mail: wanch_ma@kku.ac.th
Authors’ addresses: Tongjit Thanchomnang, Pewpan M. Intapan, Viraphong Lulitanond, Anonglak Manjai, Thidarat K. Prasongdee, and Wanchai Maleewong, Departments of Parasitology and Microbiology, Faculty of Medicine and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University, Khon Kaen 40002, Thailand. Wej Choochote, Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand.
Acknowledgment: The authors thank Dr Mark Roselieb for assistance in the manuscript preparation.
Financial support: This study was supported by the Diagnostic and Research Center for Emerging Infectious Diseases and by Khon Kaen University grants.
REFERENCES
- 1↑
World Health Organization, 1992. Lymphatic filariasis: the disease and its control. Fifth report of the WHO Expert Committee on Filariasis. World Health Organ Tech Rep Ser 821 :1–71.
- 2↑
Weil GJ, Ramzy RM, 2007. Diagnostic tools for filariasis elimination programs. Trends Parasitol 23 :78–82.
- 3↑
Rao RU, Weil GJ, Fischer K, Supali T, Fischer P, 2006. Detection of Brugia parasite DNA in human blood by real-time PCR. J Clin Microbiol 44 :3887–3893.
- 4↑
Williams SA, Laney SJ, Bierwert LA, Saunders LJ, Boakye DA, Fischer P, Goodman D, Helmy H, Hoti SL, Vasuki V, Lammie PJ, Plichart C, Ramzy RM, Ottesen EA, 2002. Development and standardization of a rapid, PCR-based method for the detection of Wuchereria bancrofti in mosquitoes, for xenomonitoring the human prevalence of bancroftian filariasis. Ann Trop Med Parasitol 96 (Suppl):S41–S46.
- 5
McCarthy JS, Zhong M, Gopinath R, Ottesen EA, Williams SA, Nutman TB, 1996. Evaluation of a polymerase chain reaction-based assay for diagnosis of Wuchereria bancrofti infection. J Infect Dis 173 :1510–1514.
- 6↑
Ramzy RM, Farid HA, Kamal IH, Ibrahim GH, Morsy ZS, Faris R, Weil GJ, Williams SA, Gad AM, 1997. A polymerase chain reaction-based assay for detection of Wuchereria bancrofti in human blood and Culex pipiens. Trans R Soc Trop Med Hyg 91 :156–160.
- 7↑
Zarlenga DS, Higgins J, 2001. PCR as a diagnostic and quantitative technique in veterinary parasitology. Vet Parasitol 101 :215–230.
- 8↑
Menard A, Dachet F, Prouzet-Mauleon V, Oleastro M, Megraud F, 2005. Development of a real-time fluorescence resonance energy transfer PCR to identify the main pathogenic Campylobacter spp. Clin Microbiol Infect 11 :281–287.
- 9
Hakhverdyan M, Rasmussen TB, Thoren P, Uttenthal A, Belak S, 2006. Development of a real-time PCR assay based on primer-probe energy transfer for the detection of swine vesicular disease virus. Arch Virol 151 :2365–2376.
- 10↑
Abdelbaqi K, Buissonniere A, Prouzet-Mauleon V, Gresser J, Wesley I, Megraud F, Menard A, 2007. Development of a real-time fluorescence resonance energy transfer PCR to detect arcobacter species. J Clin Microbiol 45 :3015–3021.
- 11↑
Walker NJ, 2001. Real-time and quantitative PCR: applications to mechanism-based toxicology. J Biochem Mol Toxicol 15 :121–127.
- 12↑
Rao RU, Atkinson LJ, Ramzy RM, Helmy H, Farid HA, Bockarie MJ, Susapu M, Laney SJ, Williams SA, Weil GJ, 2006. A real-time PCR-based assay for detection of Wuchereria bancrofti DNA in blood and mosquitoes. Am J Trop Med Hyg 74 :826–832.
- 13↑
Fischer P, Erickson SM, Fischer K, Fuchs JF, Rao RU, Christensen BM, Weil GJ, 2007. Persistence of Brugia malayi DNA in vector and non-vector mosquitoes: implications for xenomonitoring and transmission monitoring of lymphatic filariasis. Am J Trop Med Hyg 76 :502–507.
- 14↑
Lulitanond V, Intapan PM, Pipitgool V, Choochote W, Maleewong W, 2004. Rapid detection of Wuchereria bancrofti in mosquitoes by LightCycler polymerase chain reaction and melting curve analysis. Parasitol Res 94 :337–341.
- 15↑
McReynolds LA, DeSimone SM, Williams SA, 1986. Cloning and comparison of repeated DNA sequences from the human filarial parasite Brugia malayi and the animal parasite Brugia pahangi. Proc Natl Acad Sci U S A 83 :797–801.
- 16↑
Choochote W, Keha P, Sukhavat K, Khamboonruang C, Sukontason K, 1987. Aedes (Finlaya) togoi Theobald 1907, Chanthaburi strain, a laboratory vector in studies of filariasis in Thailand. Southeast Asian J Trop Med Public Health 18 :259–260.
- 17↑
Maleewong W, Choochote W, Sukhavat K, Khamboonruang C, Arunyanart C, 1987. Scanning electron microscopic study of third-stage larva of Wuchereria bancrofti and Brugia malayi in Thailand. Southeast Asian J Trop Med Public Health 18 :261–264.
- 18↑
Chomcharn Y, Surathin K, Bunnag D, Sucharit S, Harinasuta T, 1980. Effect of a single dose of primaquine on a Thai strain of Plasmodium falciparum. Southeast Asian J Trop Med Public Health 18 :408–412.
- 19↑
Ghedin E, Wang S, Foster JM, Slatko BE, 2004. First sequenced genome of a parasitic nematode. Trends Parasitol 20 :151–153.
- 20↑
Fischer P, Wibowo H, Pischke S, Ruckert P, Liebau E, Ismid IS, Supali T, 2002. PCR-based detection and identification of the filarial parasite Brugia timori from Alor Island, Indonesia. Ann Trop Med Parasitol 96 :809–821.
- 21↑
Fischer P, Boakye D, Hamburger J, 2003. Polymerase chain reaction-based detection of lymphatic filariasis. Med Microbiol Immunol (Berl) 192 :3–7.