Single-step PCR Research Articles

Validation of an apicoplast genome target for the detection of Plasmodium species using polymerase chain reaction and loop mediated isothermal amplification

The genome of the Plasmodium apicoplast, which has a higher copy number compared with current targets for molecular diagnosis of malaria, appears to be a suitable target for detection of submicroscopic infections that are capable of sustaining transmission. Novel primers targeting a conserved segment of the apicoplast (PFC10_AP|0010:rRNA) were designed and used in a number of different high throughput platforms such as single-step PCR (ssPCR), nested PCR (nPCR) and loop-mediated isothermal amplification (LAMP) for parasite detection. Replicates of ten-fold serial dilutions of Plasmodium falciparum 3D7 DNA, with equivalent parasite density ranges of 200 000 to 0.2 parasites/μL, were used to determine the limit of detection and repeatability of each assay. A panel of 184 archived DNA samples extracted from either EDTA whole blood or dried blood spots, from across West Africa and South East Asia was used to determine the diagnostic performance of the assays. All assays amplified the 2 parasites/μL dilution except the ssPCR, which amplified two of the three replicates. Using an 18S rRNA PCR as reference, the sensitivity was 98% (95% CI 93–100%) for the LAMP assay, 87% (95% CI 79–93%) for ssPCR and 100% (95% CI 97–100%) for nPCR. Specificity was 91% (95% CI 83–96%) for LAMP, 82% (95% CI 72–90%) for ssPCR and 66% (95% CI 54–76%) for nPCR. The apicoplast genome-based nPCR detected more positive samples overall than the reference method. Discrepant samples were confirmed as true positives using a probe-based real-time quantitative PCR assay. The results show that the apicoplast genome is a suitable target for molecular diagnosis of malaria.

The role of adenovirus 36 as a risk factor in obesity: The first clinical study made in the fatty tissues of adults in Turkey

Obesity which developes due to multifactorial reasons, was associated recently with human Adenovirus-36 (Ad-36). The aim of this study was to investigate the prevalence of Ad-36 antibodies in obese adults and also to investigate the DNA of Ad-36 in their adipose tissue. In this cross-sectional and case-control based study, 49 obese adults, with BMI ≥30 kg/m2, and 49 non-obese adults, with BMI ≤25 kg/m2, applied for esthetic purposes and were included in this study as patient and control groups, respectively. Adipose tissue samples, obtained by the lipoaspiration method, were studied by single-step PCR and nested-PCR methods. Simultaneously, the presence of Ad-36 antibodies and serum leptin and adiponectin levels were assessed by serum neutralization assay (SNA) and ELISA, respectively. Serum samples which didn't cause a cytopathic effect at ≥1:8 were accepted as positive. Ad-36 antibody was detected in 6 (12.2%) of 49 patients by SNA and was statistically significant (p < 0.05). Ad-36 DNA was not detected in any of the adipose tissue samples of the patient or control groups. Mean BMI and leptin levels were higher in the Ad-36-positive group, while adiponectin levels were found to be lower in the Ad-36-positive group. Although no statistically significant difference was found in cholesterol and triglyceride levels between the two groups (p > 0.05), lower mean serum cholesterol and triglyceride levels were found in the Ad-36-positive patients. In conclusion, we couldn't detect Ad-36 DNA in adipose tissue; however, we detected significantly higher Ad-36 antibody levels in the obese group compared to the non-obese group, according to the both univariant and multivariant analyses, suggesting that Ad-36 may play a role in obesity. There is a need for new and extended serial, particularly cohort and human-based, studies in order to have a clear understanding of the Ad-36-obesity relationship.

Porphyromonas gingivalis in periodontal pockets and heart valves

Background There is evidence that advanced infectious chronic periodontal inflammatory disease may have an impact on general health including cardiovascular diseases. The aim of this clinical study was to evaluate the ability of Porphyromonas gingivalis to colonize heart valves and, subsequently, to assess whether there is an association between the presence of the DNA of Porphyromonas gingivalis in periodontal pockets and in degenerated heart valves.Materials and Methods Thirty patients were enrolled in the study and 31 valve specimens harvested during cardiac surgery operations were examined. All patients underwent a periodontal examination. To evaluate the periodontal status of the patients the following clinical parameters were recorded: the pocket depth, bleeding on probing (BOP) and aproximal plaque index (API). The presence of P. gingivalis in heart valve specimens and samples from periodontal pockets was analyzed using a single–step PCR method.ResultsP. gingivalis DNA was detected in periodontal pockets of 15 patients (50%). However, the DNA of this periopathogen was found neither in the aortic nor in the mitral valve specimens.Conclusions This study suggests that P. gingivalis may not have an influence on the development of the degeneration of aortic and mitral valves.

PCR-Mediated Site-Directed Mutagenesis

Unlike traditional site-directed mutagenesis, this protocol requires only a single PCR step using full plasmid amplification to generate point mutants. The method can introduce small mutations into promoter sites and is even better suited for introducing single or double mutations into proteins. It is elegant in its simplicity and can be applied quite easily in any laboratory using standard protein expression vectors and commercially available reagents.

Murine Typhus in Humans, Yucatan, Mexico

To the Editor: Rickettsia typhi is the causal agent of murine typhus, a febrile illness affecting humans worldwide (1). In Mexico, recent studies demonstrated a 14% prevalence of antibodies against typhus group rickettsiae in healthy adult blood donors in Mexico City, and a recent nonfatal case of endemic typhus was reported in Yucatan (2,3). In May 2011, a 42-year-old woman and her 12-year-old son sought care at the clinical service of the Autonomous University of Yucatan. They had malaise, headache, fever (39°C), fibromyalgia, sore throat, and fatigue and an erythematous rash on the chest that after 6 days spread to the abdomen and extremities. Dengue fever was diagnosed, and the patients were treated empirically with acyclovir, methanesulfonamide, N-(4-nitro-2-phenoxyphenyl) and clarithromycin. Dengue could not be confirmed by laboratory testing. Murine typhus was diagnosed on the basis of PCR amplification and immunofluorescent assay for antibodies to R. typhi. Rickettsia species was determined by sequencing of rickettsial genes. Three serum samples were collected from the woman (8, 12, and 16 days after illness onset) and 1 from the boy (8 days) in 3.8% sodium citrate as anticoagulant, and DNA was extracted immediately by QIAamp DNA Blood Mini Kit (QIAGEN Valencia, CA, USA) in accordance with the manufacturer’s instructions. Single-step PCR amplification was performed by using genus-specific primers for the rickettsial 17-kDa protein and citrate synthase (gltA) genes as reported (4) Sequences of the citrate synthase and 17-kDa PCR products were compared at the National Center for Biotechnology Information BLAST software (5). Three PCR amplicons of both genes were fully sequenced and compared with sequences in GenBank. The 17-kDa and citrate synthase fragment sequences (GenBank accession nos. {type:entrez-nucleotide,attrs:{text:JX198507,term_id:402230109}}JX198507 and {type:entrez-nucleotide,attrs:{text:JX458814,term_id:406654549}}JX458814) showed 99% and 100% identity, respectively, with R. typhi strain Wilmington strain (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AE017197.1,term_id:51459527}}AE017197.1) (Table). Table Ig detection by immunofluorescent assay and molecular results by PCR and sequence identity of the amplicons of gltA and 17-kDa genes of Rickettsia spp. from 2 patients with murine typhus, Yucatan, Mexico Immunofluorescent assay was performed by using R. rickettsii and R. typhi antigen fixed on slides. We examined the serum samples for IgG and IgM, assessing reactivity of γ chain–specific and μ heavy chain–specific secondary conjugates, respectively, with rickettsial antigens. All 3 samples from the woman and the sample from the boy contained antibodies to R. typhi (Table). Both patients were treated with 100 mg of oral doxycycline 2×/day for 7 days (boy), and 10 days (woman); symptoms improved in 72 hours for the child. The woman’s symptoms resolved completely in 5 days. Typhus has been endemic in Mexico since before the conquest period (6). Socioeconomic aspects play a major role in zoonotic diseases, such as rickettsioses, especially in their distribution in urban and suburban areas because of factors such as marginalized communities, animal breeding, education levels, poverty, and social exclusion from health systems. Overcrowding resulting from migration from rural areas to large urban centers contributes to increased zoonoses in urban areas. Also contributing is the ecologic imbalance of flora and fauna associated with deteriorating sanitary conditions in areas where mammals involved in the cycle of R. typhi, such as rodents and opossum, may live in the same habitat as humans and colonize backyards, waste deposit area, and areas around the neighborhoods where they can find food. The concurrence and presence of mammals, vectors, and humans may contribute to maintaining transmission of endemic typhus in a reduced area, with the possibility to cause outbreaks (7,8). Housing conditions and culture, such as courtyards with vegetation and presence of pets, in several suburban areas of Yucatan encourage close contact between humans and possible reservoirs for several infectious diseases, such as mice, rats, opossums, dogs and cats, and their ectoparasites (9). Because housing and cultural conditions are similar in all countries of Latin America, endemic typhus probably is transmitted in the same way: by coexistence with domestic animals and close contact with wild reservoirs. Epidemiologic control should be closely linked to education aimed at encouraging villagers to interrupt the cycle of transmission and thus prevent the disease. We have confirmed the presence of murine typhus in Yucatan. The finding of human cases demonstrates the need to consider R. typhi infection in the differential diagnosis of febrile illnesses considered endemic in Yucatan, such as dengue fever and leptospirosis (10). Early and accurate diagnosis should enable physicians to treat this disease appropriately and early in the clinical course to prevent increased illness and death.

Tagging the Expressed Protein with 6 Histidines: Rapid Cloning of an Amplicon with Three Options

We report the designing of three expression vectors that can be used for rapid cloning of any blunt-end DNA segment. Only a single set of oligonucleotides are required to perform the amplification of the target DNA and its cloning in all three vectors simultaneously. The DNA thus cloned can express a protein either with or without a hexa-histidine tag depending upon the vector used. The expression occurs from T7 promoter when transformed into E. coli BL21(DE3). Two of the three plasmids have been designed to provide the expressed protein with either N- or C-terminus 6 histidine amino acids in tandem. The third plasmid, however, does not add any tag to the expressed protein. The cloning is achieved quickly with the requirement of phosphorylation of PCR product without any restriction digestion. Additionally, the generated clones can be confirmed with a single step PCR reaction carried out from bacterial colonies (generally termed as “colony PCR”). We show the cloning, expression and purification of Green Fluorescent Protein (GFP) as proof-of-concept. Additionally, we also show the cloning and expression of four sigma factors from Mycobacterium tuberculosis further demonstrating the utility of the designed plasmids. We strongly believe that the vectors and the strategy that we have developed will facilitate the rapid cloning and expression of any gene in E. coli BL21(DE3) with or without a hexa-histidine tag.

Multiplex DNA amplification and barcoding in a single reaction for 454 Roche sequencing: A comprehensive study on the control region of the mitochondrial genome

The analysis of the non-coding region of the mitochondrial genome using Sanger sequencing remains a laborious and time-consuming assay with too low resolution for the identification of low-frequency heteroplasmy or for mixture interpretation. In this study, an experimental design was tested in which the complete hypervariable region of the mitochondrial genome was sequenced using a novel barcoding strategy. The strategy involves a single-step multiplex nested PCR and we demonstrate its effectiveness by sequencing two multiplex reactions of two amplicons each covering the complete hypervariable region of the mitochondrial genome for 58 reference samples, 30 of which were analysed in triplicate, and 10 casework samples, each analysed in triplicate, on a 454 Roche DNA pyrosequencer with GS FLX chemistry using Multiplex Identifier (MID) primers to discriminate between samples. The generated reads for forensic (±3600 reads/MID) and reference samples (±466 reads/MID) allowed us to evaluate the accuracy in SNP calling and the variation in heteroplasmy and sequencing error rates in homopolymeric stretches between replicates.

A single-step polymerase chain reaction for simultaneous detection and differentiation of nontypeable and serotypeable Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae

ObjectiveThe critically high prevalence of bacterial otitis media worldwide has prompted a proper disease management. While vaccine development for otitis media is promising, the reliable and effective methods for diagnosis of such etiologic agents are of importance. MethodsWe developed a multiplex polymerase chain reaction assay for simultaneous detection and differentiation of nontypeable and serotypeable Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae. Five primer pairs targeting genes fumarate reductase (H. influenzae), outer membrane protein B (M. catarrhalis), major autolysin (S. pneumoniae), capsulation-associated BexA protein (all encapsulated H. influenzae) and 16S rRNA were incorporated in this single-step PCR. Validation of the multiplex PCR was also performed on clinical isolates. ResultsThe developed multiplex PCR was highly specific, enabling the detection of the target pathogens in a specific manner, either individually or as a mixture of all target organisms. The assay was also found to be sensitive with the lowest detection limit of 1ng of bacterial DNA. When applied to clinical isolates from diverse specimen sources, the multiplex PCR developed in this study correctly identified each microorganism individually or in a combination of two or more target organisms. All results matched with conventional culture identification. In addition, the ability of such assay to differentiate H. influenzae encapsulation from the study clinical isolates was 100%. ConclusionOur multiplex PCR provides a rapid and accurate diagnostic tool for detection of the 4 target organisms. Such assay would serve as a useful tool for clinicians and epidemiologists in their efforts to the proper treatment and disease management caused by these organisms.

Multiple tick-associated bacteria in Ixodes ricinus from Slovakia

Eighty Ixodes ricinus ticks collected in the Rovinka forest in Slovakia were tested by real-time and routine single-step PCR for the presence of different pathogenic and endosymbiotic bacteria. No evidence of Coxiella burnetii, Diplorickettsia massiliensis, or Bartonella spp. was found. However, we identified, as the pathogenic bacteria, Anaplasma phagocytophilum in 8%, Rickettsia helvetica in 11%, Borrelia afzelii in 9%, Bo. garinii in 8%, Bo. burgdorferi sensu stricto in 5%, Bo. valaisiana in 3%, and Bo. miyamotoi in 1% of ticks. Other bacteria with unknown or suspected pathogenicity for humans were also identified: Arsenophonus nasoniae in 25%, Spiroplasma ixodetis in 3%, Candidatus Midichloria mitochondrii in 4%, Wolbachia aff. pipientis in 14%, and Ehrlichia muris in 3% of ticks. A. nasoniae was found almost exclusively in nymphs, and E. muris was only found in adult ticks. In total, 65% of ticks contained at least one species of bacteria, and 16% contained 2 or more species.

Intracellular Trafficking of FXYD1 (Phospholemman) and FXYD7 Proteins in Xenopus Oocytes and Mammalian Cells

FXYD proteins are a group of short single-span transmembrane proteins that interact with the Na(+)/K(+) ATPase and modulate its kinetic properties. This study characterizes intracellular trafficking of two FXYD family members, FXYD1 (phospholemman (PLM)) and FXYD7. Surface expression of PLM in Xenopus oocytes requires coexpression with the Na(+)/K(+) ATPase. On the other hand, the Na(+)/Ca(2+) exchanger, another PLM-interacting protein could not drive it to the cell surface. The Na(+)/K(+) ATPase-dependent surface expression of PLM could be facilitated by either a phosphorylation-mimicking mutation at Thr-69 or a truncation of three terminal arginine residues. Unlike PLM, FXYD7 could translocate to the cell surface of Xenopus oocytes independently of the coexpression of α1β1 Na(+)/K(+) ATPase. The Na(+)/K(+) ATPase-independent membrane translocation of FXYD7 requires O-glycosylation of at least two of three conserved threonines in its ectodomain. Subsequent experiments in mammalian cells confirmed the role of conserved extracellular threonine residues and demonstrated that FXYD7 protein, in which these have been mutated to alanine, is trapped in the endoplasmic reticulum and Golgi apparatus.

A novel PCR-based method for high throughput prokaryotic expression of antimicrobial peptide genes

BackgroundTo facilitate the screening of large quantities of new antimicrobial peptides (AMPs), we describe a cost-effective method for high throughput prokaryotic expression of AMPs. EDDIE, an autoproteolytic mutant of the N-terminal autoprotease, Npro, from classical swine fever virus, was selected as a fusion protein partner. The expression system was used for high-level expression of six antimicrobial peptides with different sizes: Bombinin-like peptide 7, Temporin G, hexapeptide, Combi-1, human Histatin 9, and human Histatin 6. These expressed AMPs were purified and evaluated for antimicrobial activity.ResultsTwo or four primers were used to synthesize each AMP gene in a single step PCR. Each synthetic gene was then cloned into the pET30a/His-EDDIE-GFP vector via an in vivo recombination strategy. Each AMP was then expressed as an Npro fusion protein in Escherichia coli. The expressed fusion proteins existed as inclusion bodies in the cytoplasm and the expression levels of the six AMPs reached up to 40% of the total cell protein content. On in vitro refolding, the fusion AMPs was released from the C-terminal end of the autoprotease by self-cleavage, leaving AMPs with an authentic N terminus. The released fusion partner was easily purified by Ni-NTA chromatography. All recombinant AMPs displayed expected antimicrobial activity against E. coli, Micrococcus luteus and S. cerevisia.ConclusionsThe method described in this report allows the fast synthesis of genes that are optimized for over-expression in E. coli and for the production of sufficiently large amounts of peptides for functional and structural characterization. The Npro partner system, without the need for chemical or enzymatic removal of the fusion tag, is a low-cost, efficient way of producing AMPs for characterization. The cloning method, combined with bioinformatic analyses from genome and EST sequence data, will also be useful for screening new AMPs. Plasmid pET30a/His-EDDIE-GFP also provides green/white colony selection for high-throughput recombinant AMP cloning.

Genotyping the CRHR1 rs242939 (A&gt;G) Polymorphism by a One-Step Tetra Primer–Amplification Refractory Mutation System–Polymerase Chain Reaction

With the rapid advances in molecular techniques, various methods for genotyping single-nucleotide polymorphisms (SNPs) are available. Still, the search for easy, robust, and less costly techniques continues. We wished to develop a Tetra primer-amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) based technique for the corticotrophin-releasing hormone receptor 1 (CRHR1) (rs242939) SNP for use in our research lab. To detect SNPs in a single-step PCR, we set up two genotyping methods, T-ARMS-PCR and restriction fragment length polymorphism (RFLP). The study was performed using thirty blood samples taken from clinically defined asthmatic patients. The efficiency and effectiveness of results obtained by T-ARMS were validated by PCR-RFLP and sequencing. This study demonstrates that T-ARMS is feasible and applicable to discriminate a wild-type allele from the respective mutant allele in one step. This work is the first that presents a rapid, sensitive, and high throughput genotyping method for the CRHR1 (rs242939) polymorphism and can be used for both large- and small-scale genotyping studies.

A New Single-Step PCR Assay for the Detection of the Zoonotic Malaria Parasite Plasmodium knowlesi

BackgroundRecent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection.Methodology and Significant FindingsWe have developed a bioinformatics approach to search the available malaria parasite genome database for the identification of suitable DNA sequences relevant for molecular diagnostic tests. Using this approach, we have identified multi-copy DNA sequences distributed in the P. knowlesi genome. We designed and tested several novel primers specific to new target sequences in a single-tube, non-nested PCR assay and identified one set of primers that accurately detects P. knowlesi. We show that this primer set has 100% specificity for the detection of P. knowlesi using three different strains (Nuri, H, and Hackeri), and one human case of malaria caused by P. knowlesi. This test did not show cross reactivity with any of the four human malaria parasite species including 11 different strains of P. vivax as well as 5 additional species of simian malaria parasites.ConclusionsThe new PCR assay based on novel P. knowlesi genomic sequence targets was able to accurately detect P. knowlesi. Additional laboratory and field-based testing of this assay will be necessary to further validate its utility for clinical diagnosis of P. knowlesi.

Effects of Decontamination, DNA Extraction, and Amplification Procedures on the Molecular Diagnosis of Mycobacterium ulcerans Disease (Buruli Ulcer)

We compared two DNA extraction methods (a semiautomated method using a Maxwell kit and a modified Boom method) and three amplification procedures (a single-step PCR, a nested PCR, and a real-time quantitative PCR) on 74 surgical tissue specimens from patients with clinically suspected Buruli ulcer. All of these procedures were compared before and after decontamination. We observed that, among the procedures tested, real-time PCR after the modified Boom extraction method or a single-run PCR assay after the Maxwell 16 extraction method, performed on nondecontaminated suspensions, are the best for the molecular diagnosis of Mycobacterium ulcerans disease.

A rapid and highly reliable field-based LAMP assay of canine parvovirus

Loop-mediated isothermal amplification (LAMP) is known as a rapid and reliable alternative to conventional single-step or nested PCR for detection of genomic DNA of various pathogens in clinical samples. In this study, LAMP assay was developed for canine parvovirus (CPV) and compared with single-step and nested PCR assays. Out of 50 fecal samples from dogs clinically suspected for CPV infections, 19 were found positive by single-step PCR, 22 by nested PCR and 26 by LAMP. LAMP products were subjected to restriction analysis and sequencing to check their specificity. LAMP assay turned out to be a rapid and fairly reproducible method, did not amplify other common canine pathogens and was more sensitive than nested PCR assay. Therefore, it can be regarded as a highly reliable method for routine field diagnosis of CPV infection. canine parvovirus; nested polymerase chain reaction; loop-mediated isothermal amplification; sensitivity; specificity.

Novel genetic tools for Hansenula polymorpha

Hansenula polymorpha is an important yeast in industrial biotechnology. In addition, it is extensively used in fundamental research devoted to unravel the principles of peroxisome biology and nitrate assimilation. Here we present an overview of key components of the genetic toolbox for H.polymorpha. In addition, we present new selection markers that we recently implemented in H. polymorpha. We describe novel strategies for the efficient creation of targeted gene deletions and integrations in H.polymorpha. For this, we generated a yku80 mutant, deficient in non-homologous end joining, resulting in strongly enhanced efficiency of gene targeting relative to the parental strain. Finally, we show the implementation of Gateway technology and a single-step PCR strategy to create deletions in H.polymorpha.

Development of a Multiplex PCR for Identification of Vineyard Mealybugs

A simple molecular tool was developed and tested to identify seven mealybug species found in North American vineyards: Pseudococcus maritimus Ehrhorn, Pseudococcus viburni (Signoret), Pseudococcus longispinus (Targioni-Tozzeti), Pseudococcus calceolariae (Maskell), Planococcus ficus (Signoret), Planococcus citri (Risso), and Ferrisia gilli Gullan. The developed multiplex PCR is based on the mitochondrial cytochrome c oxidase subunit one gene. In tests, this single-step multiplex PCR correctly identified 95 of 95 mealybug samples, representing all seven species and collected from diverse geographic regions. To test the sensitivity, single specimen samples with different Pl. ficus developmental stages (egg to adult female and adult male) were processed PCR and the resulting output provided consistent positive identification. To test the utility of this protocol for adult males caught in sex baited pheromone traps, Pl. ficus adult males were placed in pheromone traps, aged at a constant temperature of 26±2°C, and processed with the multiplex each day thereafter for 8 d. Results showed consistent positive identification for up to 6 d (range, 6-8 d). Results are discussed with respect to the usefulness of this molecular tool for the identification of mealybugs in pest management programs and biosecurity of invasive mealybugs.

Development and evaluation of a single‐step multiplex PCR to differentiate the aquatic stages of morphologically similar Aedes (subgenus: Stegomyia) species

To develop a single-step multiplex PCR to differentiate the aquatic stages of Aedes aegypti, Aedes albopictus and Aedes vittatus collected from different breeding spots in arbovirus endemic/epidemic areas and to detect the most abundant species by the multiplex PCR. Aquatic stages of different mosquito species were sampled by inspecting artificial and natural breeding sites in domestic and peridomestic areas. DNA was isolated from different stages of the three Aedes species. Using novel primers based on 18S rDNA sequence, a single-step multiplex PCR was developed to clearly distinguish the three Aedes species. It was then evaluated in the aquatic stages of Aedes species collected from different areas. A total of 1150 aquatic stages were collected from 294 breeding spots, of which 156 contained Aedes species. Discarded tires were the major breeding spots of Aedes species. The aquatic stages were clustered into 230 pools; Ae. albopictus was detected in the largest number of pools, followed by Ae. aegypti and Ae. vittatus. The Multiplex PCR clearly differentiated the aquatic stages of the three Aedes species and detected that Ae. albopictus was most profuse in different breeding spots surveyed, hence indicating to be the main vector in this region. So control measures can be designed against Ae. albopictus at an early stage to prevent any arboviral outbreak. This method is a convenient tool for precise identification of Aedes vectors during entomological surveys in arbovirus endemic/epidemic areas where several species coexist.

Specific primers for PCR amplification of the ITS1 (ribosomal DNA) of Trypanosoma lewisi

Trypanosoma lewisi is a mild or non-pathogenic parasite of the sub-genus Herpetosoma transmitted by fleas to rats. In a previous study we described pan-trypanosome specific primers TRYP1 which amplify the ITS1 of ribosomal DNA by hybridizing in highly conserved regions of 18S and 5.8S genes. These primers proved to be useful for detecting T. lewisi DNA in laboratory rats, but a recent large scale survey in wild rodents demonstrated a lack of specificity. In the present study, we designed and evaluated mono-specific primers LEW1S and LEW1R, for the detection and identification of T. lewisi by a single-step PCR. These primers were designed inside the highly variable region of the ITS1 sequence of T. lewisi ribosomal DNA. The product size of 220bp is specific to T. lewisi. The sensitivity limit was estimated between 0.055 and 0.55pg of DNA per reaction, equivalent to 1–10 organisms per reaction. All the PCR products obtained from 6 different T. lewisi isolates were more than 98% similar with each other and similar to the sequences of T. lewisi already published in Genbank. All DNA of 7 T. lewisi stocks from China gave the specific 220bp product. We showed that LEW1S and LEW1R primers enabled sensitive detection and identification of T. lewisi infection in laboratory and wild rats. This assay is recommended for monitoring T. lewisi infections in rat colonies or for studying infections in the wild fauna. An absence of cross reaction with human DNA means that these primers can be used to investigate atypical trypanosome infections in humans. Given the risk of T. lewisi infection in human, we believe that these primers will be beneficial for public health diagnosis and rodents investigation programmes.

Applied Genomics: Data Mining Reveals Species-Specific Malaria Diagnostic Targets More Sensitive than 18S rRNA

Accurate and rapid diagnosis of malaria infections is crucial for implementing species-appropriate treatment and saving lives. Molecular diagnostic tools are the most accurate and sensitive method of detecting Plasmodium, differentiating between Plasmodium species, and detecting subclinical infections. Despite available whole-genome sequence data for Plasmodium falciparum and P. vivax, the majority of PCR-based methods still rely on the 18S rRNA gene targets. Historically, this gene has served as the best target for diagnostic assays. However, it is limited in its ability to detect mixed infections in multiplex assay platforms without the use of nested PCR. New diagnostic targets are needed. Ideal targets will be species specific, highly sensitive, and amenable to both single-step and multiplex PCRs. We have mined the genomes of P. falciparum and P. vivax to identify species-specific, repetitive sequences that serve as new PCR targets for the detection of malaria. We show that these targets (Pvr47 and Pfr364) exist in 14 to 41 copies and are more sensitive than 18S rRNA when utilized in a single-step PCR. Parasites are routinely detected at levels of 1 to 10 parasites/μl. The reaction can be multiplexed to detect both species in a single reaction. We have examined 7 P. falciparum strains and 91 P. falciparum clinical isolates from Tanzania and 10 P. vivax strains and 96 P. vivax clinical isolates from Venezuela, and we have verified a sensitivity and specificity of ∼100% for both targets compared with a nested 18S rRNA approach. We show that bioinformatics approaches can be successfully applied to identify novel diagnostic targets and improve molecular methods for pathogen detection. These novel targets provide a powerful alternative molecular diagnostic method for the detection of P. falciparum and P. vivax in conventional or multiplex PCR platforms.