Real-time fluorescence-based PCR for detection of malaria parasites.
Ontology highlight
ABSTRACT:
SUBMITTER: Lee MA
PROVIDER: S-EPMC139664 | biostudies-literature | 2002 Nov
REPOSITORIES: biostudies-literature
Publications
Real-time fluorescence-based PCR for detection of malaria parasites.
Lee May-Ann MA Tan Chiew-Hoon CH Aw Lay-Tin LT Tang Choon-Siang CS Singh Mulkit M Lee Sze-Hee SH Chia Hwee-Pin HP Yap Eric P H EP
Journal of clinical microbiology 20021101 11
A fluorescence-based real-time 5' nuclease PCR capable of detecting all four human malaria parasites was developed to screen large numbers of samples during an outbreak to prevent further transmission of malaria. The effectiveness of antimalarial therapy for malaria patients can be monitored by determining the reduction of parasitemia by this method. ...[more]
Similar Datasets
Project description:Chlamydia pneumoniae is an important respiratory pathogen recently associated with atherosclerosis and several other chronic diseases. Detection of C. pneumoniae is inconsistent, and standardized PCR assays are needed. Two real-time PCR assays specific for C. pneumoniae were developed by using the fluorescent dye-labeled TaqMan probe-based system. Oligonucleotide primers and probes were designed to target two variable domains of the ompA gene, VD2 and VD4. The limit of detection for each of the two PCR assays was 0.001 inclusion-forming unit. Thirty-nine C. pneumoniae isolates obtained from widely distributed geographical areas were amplified by the VD2 and VD4 assays, producing the expected 108- and 125-bp amplification products, respectively. None of the C. trachomatis serovars, C. psittaci strains, other organisms, or human DNAs tested were amplified. The amplification results of the newly developed assays were compared to the results of culturing and two nested PCR assays, targeting the 16S rRNA and ompA genes. The assays were compared by testing C. pneumoniae purified elementary bodies, animal tissues, 228 peripheral blood mononuclear cell (PBMC) specimens, and 179 oropharyngeal (OP) swab specimens obtained from ischemic stroke patients or matched controls. The real-time VD4 assay and one nested PCR each detected C. pneumoniae in a single, but different, PBMC specimen. Eleven of 179 OP specimens (6.1%) showed evidence of the presence of C. pneumoniae in one or more tests. The real-time VD4 assay detected the most positive results of the five assays. We believe that this real-time PCR assay offers advantages over nested PCR assays and may improve the detection of C. pneumoniae in clinical specimens.
| S-EPMC153405 | biostudies-literature
Project description:BackgroundReal-time PCR has recently become the technique of choice for absolute and relative nucleic acid quantification. The gold standard quantification method in real-time PCR assumes that the compared samples have similar PCR efficiency. However, many factors present in biological samples affect PCR kinetic, confounding quantification analysis. In this work we propose a new strategy to detect outlier samples, called SOD.ResultsRichards function was fitted on fluorescence readings to parameterize the amplification curves. There was not a significant correlation between calculated amplification parameters (plateau, slope and y-coordinate of the inflection point) and the Log of input DNA demonstrating that this approach can be used to achieve a "fingerprint" for each amplification curve. To identify the outlier runs, the calculated parameters of each unknown sample were compared to those of the standard samples. When a significant underestimation of starting DNA molecules was found, due to the presence of biological inhibitors such as tannic acid, IgG or quercitin, SOD efficiently marked these amplification profiles as outliers. SOD was subsequently compared with KOD, the current approach based on PCR efficiency estimation. The data obtained showed that SOD was more sensitive than KOD, whereas SOD and KOD were equally specific.ConclusionOur results demonstrated, for the first time, that outlier detection can be based on amplification shape instead of PCR efficiency. SOD represents an improvement in real-time PCR analysis because it decreases the variance of data thus increasing the reliability of quantification.
| S-EPMC2873533 | biostudies-literature
Project description:Reliable methods to preserve mosquito vectors for malaria studies are necessary for detecting Plasmodium parasites. In field settings, however, maintaining a cold chain of storage from the time of collection until laboratory processing, or accessing other reliable means of sample preservation is often logistically impractical or cost prohibitive. As the Plasmodium infection rate of Anopheles mosquitoes is a central component of the entomological inoculation rate and other indicators of transmission intensity, storage conditions that affect pathogen detection may bias malaria surveillance indicators. This study investigated the effect of storage time and temperature on the ability to detect Plasmodium parasites in desiccated Anopheles mosquitoes by real-time polymerase chain reaction (PCR).Laboratory-infected Anopheles stephensi mosquitoes were chloroform-killed and stored over desiccant for 0, 1, 3, and 6 months while being held at four different temperatures: 28, 37, -20 and -80°C. The detection of Plasmodium DNA was evaluated by real-time PCR amplification of a 111 base pair region of block 4 of the merozoite surface protein.Varying the storage time and temperature of desiccated mosquitoes did not impact the sensitivity of parasite detection. A two-way factorial analysis of variance suggested that storage time and temperature were not associated with a loss in the ability to detect parasites. Storage of samples at 28°C resulted in a significant increase in the ability to detect parasite DNA, though no other positive associations were observed between the experimental storage treatments and PCR amplification.Cold chain maintenance of desiccated mosquito samples is not necessary for real-time PCR detection of parasite DNA. Though field-collected mosquitoes may be subjected to variable conditions prior to molecular processing, the storage of samples over an inexpensive and logistically accessible desiccant will likely ensure accurate assessment of malaria parasite presence without diminishing PCR-detection of parasites in mosquitoes stored for at least six months.
| S-EPMC3405449 | biostudies-literature
Project description:Real-time PCR (rt-PCR) is a widely used molecular method for detection of Neisseria meningitidis (Nm). Several rt-PCR assays for Nm target the capsule transport gene, ctrA. However, over 16% of meningococcal carriage isolates lack ctrA, rendering this target gene ineffective at identification of this sub-population of meningococcal isolates. The Cu-Zn superoxide dismutase gene, sodC, is found in Nm but not in other Neisseria species. To better identify Nm, regardless of capsule genotype or expression status, a sodC-based TaqMan rt-PCR assay was developed and validated. Standard curves revealed an average lower limit of detection of 73 genomes per reaction at cycle threshold (C(t)) value of 35, with 100% average reaction efficiency and an average R(2) of 0.9925. 99.7% (624/626) of Nm isolates tested were sodC-positive, with a range of average C(t) values from 13.0 to 29.5. The mean sodC C(t) value of these Nm isolates was 17.6±2.2 (±SD). Of the 626 Nm tested, 178 were nongroupable (NG) ctrA-negative Nm isolates, and 98.9% (176/178) of these were detected by sodC rt-PCR. The assay was 100% specific, with all 244 non-Nm isolates testing negative. Of 157 clinical specimens tested, sodC detected 25/157 Nm or 4 additional specimens compared to ctrA and 24 more than culture. Among 582 carriage specimens, sodC detected Nm in 1 more than ctrA and in 4 more than culture. This sodC rt-PCR assay is a highly sensitive and specific method for detection of Nm, especially in carriage studies where many meningococcal isolates lack capsule genes.
| S-EPMC3088665 | biostudies-literature
Project description:Real-time technology eliminates many of the pitfalls of diagnostic PCR, but this method has not been applied to differentiation of Leishmania organisms so far. We have developed a real-time PCR that simultaneously detects, quantitates, and categorizes Leishmania organisms into three relevant groups causing distinct clinical pictures. The analytical sensitivity (detection rate of >or=95% at 94.1 parasites/ml of blood) was within a range that has been determined previously to facilitate the confirmation of visceral leishmaniasis from peripheral blood. Parasites were successfully detected in 12 different clinical samples (blood, bone marrow, skin, and liver). The Leishmania donovani complex, the Leishmania brasiliensis complex, and species other than these could be clearly discriminated by means of distinct melting temperatures obtained with fluorescence resonance energy transfer probes (melting points, 72.7, 67.1, and 65.0 degrees C, respectively). All three groups could be quantified within equal ranges. As in other real-time PCRs, the variability in the quantification of DNA was small (coefficient of variation [CV], <2%). However, human samples containing low levels of parasites (100 parasites per ml of blood) showed higher variation (CV, 60.89%). Therefore, despite its superior analytical performance, care must be taken when real-time PCR is utilized for therapy monitoring.
| S-EPMC153923 | biostudies-literature
Project description:IntroductionCalf diarrhea is a significant condition that has a strong effect on the cattle industry, resulting in huge economic losses annually. Bovine torovirus (BToV), bovine enterovirus (BEV), bovine norovirus (BNoV), bovine coronavirus (BCoV), bovine rotavirus (BRV), and bovine viral diarrhea virus (BVDV) are key pathogens that have been implicated in calf diarrhea. Among these viruses, there remains limited research on BToV, BEV, and BNoV, with no available vaccines or drugs for their prevention and control. Although commercial vaccines exist for BCoV, BRV, and BVDV, the prevalence of these diseases remains high.MethodsTo address this issue, we developed a multiplex real-time fluorescence quantitative PCR method for detecting BToV, BEV, BNoV, BCoV, BRV, and BVDV. This method can be used to effectively monitor the prevalence of these six viruses and serve as a reference for future prevention and control strategies. In this study, we specifically designed primers and probes for the BNoV Rdrp, BEV 5'UTR, BToV M, BCoV N, BRV NSP5, and BVDV 5'UTR genes.ResultsThis method was determined to be efficient, stable, and sensitive. The lowest detectable levels of plasmids for BNoV, BEV, BToV, BRV, BCoV, and BVDV were 1.91 copies/μL, 96.0 copies/μL, 12.8 copies/μL, 16.4 copies/μL, 18.2 copies/μL, and 65.3 copies/μL, respectively. Moreover, the coefficients of variation for all six detection methods were < 3%; they also exhibited a strong linear relationship (R2 ≥ 0.98), and an amplification efficiency of 90%-110%. A total of 295 fecal and anal swabs were collected from calves with diarrhea in Guangdong, China. The positive rates for BToV, BEV, BNoV, BCoV, BR, and BVDV were determined to be 0.34% (1/295), 6.10% (18/295), 0.68% (2/295), 1.36% (4/295), 10.85% (32/295), and 2.03% (6/295), respectively. Notably, BEV and BRV exhibited the highest prevalence.DiscussionAdditionally, this study identified the occurrence of BToV and BNoV in Guangdong for the first time. In summary, this study successfully established an effective method for detecting several important bovine viruses; ultimately, this holds strong implications for the future development of the cattle industry.
| S-EPMC10796610 | biostudies-literature
Project description:Sacbrood virus (SBV) is a picorna-like virus that affects honey bees (Apis mellifera) and results in the death of the larvae. Several procedures are available to detect Chinese SBV (CSBV) in clinical samples, but not to estimate the level of CSBV infection. The aim of this study was develop an assay for rapid detection and quantification of this virus. Primers and probes were designed that were specific for CSBV structural protein genes. A TaqMan minor groove binder (MGB) probe-based, fluorescence real-time quantitative PCR was established. The specificity, sensitivity and stability of the assay were assessed; specificity was high and there were no cross-reactivity with healthy larvae or other bee viruses. The assay was applied to detect CSBV in 37 clinical samples and its efficiency was compared with clinical diagnosis, electron microscopy observation, and conventional RT-PCR. The TaqMan MGB-based probe fluorescence real-time quantitative PCR for CSBV was more sensitive than other methods tested. This assay was a reliable, fast, and sensitive method that was used successfully to detect CSBV in clinical samples. The technology can provide a useful tool for rapid detection of CSBV. This study has established a useful protocol for CSBV testing, epidemiological investigation, and development of animal models.
| S-EPMC3568131 | biostudies-literature
Project description:BackgroundPorcine circovirus type 3 (PCV3) has been an emerging porcine virus spread around the world. The conserved DNA sequence of PCV3 enabled good performance in molecular biological assays.ResultIn this study, we developed a real-time fluorescence PCR assay for the detection of PCV3. The conserved region within Capsid genome of PCV3 was selected for the design of primer pairs and probes. After optimizing, a primer pair and probe was screened, providing high sensitivity (10 copies/μL) and specificity (no cross reaction with other porcine viruses or common bacterium). In addition, this method was applied in the detection of 110 clinical samples, and the performance was compared with other previously reported PCR and real-time PCR methods. This method provided higher detection rate.ConclusionA real-time fluorescence PCR assay has been developed for the detection of PCV3, with high sensitivity and specificity, exhibiting good performance in detecting clinical samples.
| S-EPMC7368764 | biostudies-literature
Project description:With emergence of pandemic COVID-19, rapid and accurate diagnostic testing is essential. This study compared laboratory-developed tests (LDTs) used for the detection of SARS-CoV-2 in Canadian hospital and public health laboratories, and some commercially available real-time RT-PCR assays. Overall, analytical sensitivities were equivalent between LDTs and most commercially available methods.
| S-EPMC7219382 | biostudies-literature
Project description:We developed and evaluated a real-time fluorescence PCR assay for detecting the A and B subunits of diphtheria toxin (tox) gene. When 23 toxigenic Corynebacterium diphtheriae strains, 9 nontoxigenic C. diphtheriae strains, and 44 strains representing the diversity of pathogens and normal respiratory flora were tested, this real-time PCR assay exhibited 100% sensitivity and specificity. It allowed for the detection of both subunits of the tox gene at 750 times greater sensitivity (2 CFU) than the standard PCR (1,500 CFU). When used directly on specimens collected from patients with clinical diphtheria, one or both subunits of the tox gene were detected in 34 of 36 specimens by using the real-time PCR assay; only 9 specimens were found to be positive by standard PCR. Reamplification by standard PCR and DNA sequencing of the amplification product confirmed all real-time PCR tox-positive reactions. This real-time PCR format is a more sensitive and rapid alternative to standard PCR for detection of the tox gene in clinical material.
| S-EPMC154649 | biostudies-literature