Species-specific DNA Fragments Research Articles

Development of a Multiplex PCR Assay for Rapid Differentiation of Fowlpox and Pigeonpox Viruses.

The aim of this study was to develop a multiplex PCR assay capable of rapidly differentiating two major Avipoxvirus (APV) species, Fowlpox virus (FWPV) and Pigeonpox virus (PGPV), which cause disease in bird species. Despite the importance of a rapid differentiation assay, no such assay exists that can differentiate the APV species without sequencing. To achieve this, species-specific target DNA fragments were selected from the fpv122 gene of FWPV and the HM89_gp120 gene of PGPV, which are unique to each genome. Nine samples collected from unvaccinated chickens, pigeons, and a turkey with typical pox lesions were genetically identified as FWPV and PGPV. The designed primers and target DNA fragments were validated using in silico analyses with the nucleotide Basic Local Alignment Search Tool. The multiplex PCR assay consisted of species-specific primers and previously described PanAPV primers (genus-specific) and was able to differentiate FWPV and PGPV, consistent with the phylogenetic outputs. This study represents the first successful differentiation of FWPV and PGPV genomes using a conventional multiplex PCR test. This assay has the potential to facilitate the rapid diagnosis and control of APV infections.

Accurate Diagnosis of Schistosoma mansoni and S. haematobium from Filtered Urine Samples Collected in Tanzania, Africa.

Schistosomiasis is a bloodborne, and waterborne parasitic disease caused by the human Schistosoma species, namely Schistosoma mansoni and S. haematobium. The parasite requires an intermediate snail host, where they grow and develop, along with a human host (definitive). Schistosoma egg detection in feces (S. mansoni) and urine (S. haematobium) are the WHO-recommended confirmatory diagnostic tests. The goal of our research was to determine the efficacy of detecting single or dual Schistosome species from filtered human urine samples collected in Tanzania by amplifying species-specific cell-free repeat DNA fragments via polymerase chain reaction (PCR) and gel electrophoresis. In total, 104 filtered human urine samples were evaluated and collected from individuals residing in the village of Kayenze, Tanzania. All samples were detected with 100% accuracy and no cross-amplification was present. For a single infection of S. mansoni, 22 (21%) of the samples were positive, while 15 (14%) of the samples were negative via PCR. Moreover, for a single infection of S. haematobium, 7 (7%) of the samples were positive, while 15 (14%) of the samples were negative. Dual infections were found in a higher percentage, with 60 (58%) of the samples being positive. Thus, we have justified that PCR is more sensitive and specific by amplifying species-specific cell-free repeat DNA fragments from the same urine sample than WHO-recommended methods of processing stool and urine.

Sexually Transmitted Infections in Male Patients with Urethritis.

(1) Background: Sexually Transmitted Infections (STIs) in men are a significant public health problem due to the consequences they can have, such as chronic diseases, infertility, cancer, and even death. This study aimed to determine the frequency of microorganisms associated with STIs in men with urethritis attending urology consultations, and to explore their clinical correlations. (2) Methods: A population that attended the urology consultation of the University Hospital "Dr. José E. González" was studied. Written consent was obtained, and interviews and clinical history were conducted about specific risk factors identifying signs and symptoms associated with any genitourinary condition; after that, urine samples were collected. Identification of C. trachomatis, N. gonorrhoeae, U. urealyticum, and M. genitalium was based on amplifying species-specific DNA fragments. (3) Results: A total of 200 patients were included. The mean age was 55 years (20-95). According to the interviews, only 32.5% (n = 65) had received prior sex education. Additionally, 75% (n = 150) do not usually use any protection during sexual intercourse. Regarding clinical factors, 69.4% (n = 138) presented burning or pain when urinating. Molecular analysis revealed the presence of C. trachomatis to be 9.5% (n = 19), with U. urealyticum at 13% (n = 26), and M. genitalium at 2% (n = 4). (4) Conclusions: This is the most extensive molecular epidemiological study of the frequency of STIs in men in Mexico in third-level care and its association with different risk factors. As reported globally, a similar frequency of C. trachomatis, U. urealyticum, and M. genitalium was detected.

Development of a new PCR assay and a recombinase-aided amplification based isothermal amplification coupled with lateral flow dipstick assay for potato late blight detection

Late blight caused by Phytophthora infestans is the most devastating potato disease, yet leaf infections are not reliably detected by current assays during the incubation phase of the pathogen and asymptomatic plants. In this study, the genome of P. infestans was queried against the NCBI nucleic acid database and a 1050 bp species-specific DNA fragment was identified. Using this fragment, polymerase chain reaction (PCR) and recombinase-aided amplification-lateral flow dipstick (RAA-LFD) assays were developed for the detection of potato late blight pathogen. Both assays effectively distinguished P. infestans from related species (e.g., P. capsici). The detection limits of the three PCR primer pairs under optimized conditions (59.3 °C, 35 PCR cycles) were 1.31 × 10−1 fg, 1.18 × 10−2 fg, and 1.24 × 10−1 fg, corresponding to 27.31 copies, 2.49 copies, and 25.51 copies, respectively. While the detection limit of the RAA-LFD assay at 37 °C for 25 min was 5 × 10−4 fg (0.10 copies), which was better than all reported molecular detection assays for potato late blight. Both PCR (59.3 °C, 35 PCR cycles) and RAA-LFD (37 °C for 10 min) positively detected all symptomatic leaves. When extending the amplification time to 20 min, the RAA-LFD assay detected most of the leaves with incubation phase of the disease. The molecular detection assays developed in this study were species-specific and are expected to be used in detecting potato late blight in the incubation phase in leaves and asymptomatic plants, which will aid in accurate monitoring and early warning of potato late blight.

Qualitative and Quantitative Detection of Mealworm DNA in Raw and Commercial Food Products Using Real-Time PCR

Considering food safety and an increasing public awareness of the ingredients, production process and origin of foods, the application of insects as food requires the development of tests for the reliable identification of their presence. The aim of the study was (1) the determination of appropriate modifications of the selected method for isolating the DNA of two life stages of mealworm, i.e., larva and adult, from commercial food products; (2) the determination of the method parameters for the qualitative and quantitative analysis of mealworm contents based on the detection of a species-specific mitochondrial DNA fragment, using real-time PCR; (3) the application of a method to test the commercial food products of mealworm. A total of nine species of adult insect were investigated (field cricket, Dubia cockroach, Madagascar cockroach, banded cricket, migratory locust, yellow mealworm, superworm, house fly and lacewing), theirlarvaes (yellow mealworms and superworms) and thirteen commercial food products (dried whole insects, powder and granules) representing various insect species and origins which were purchased from the European market. The obtained results showed that the efficiency of the modification of the DNA extraction method is dependent on the life stage of the mealworm. We proved the high sensitivity of the test, with the range of the method being 0.1–100%; we also proved the biological specificity in this range, and the linearity. The linearity of the test was also statistically verified using the Fisher–Snedecor test. One-way variance analysis showed statistically significant differences between the cT values of the two mealworm life stages studied, and similarly, between the threshold cycle (cT) values of adult forms. In contrast, for the inside group of mealworm larvae, there was no significant difference observed between the results of the cT values. The test is effective for processed food products and may be used to monitor food. The research proved the suitability of the applied method for the analysis of samples that are commercially available as food for exotic animals. The hereby-developed method is based on widely used laboratory techniques, and does not require any additional investment in equipment. The availabilityof such a methodallows for the verification of the accuracy of the declared species component of the food products.

Molecular diversity and phylogenetic analysis of Azerbaijan oaks (Quercus spp.) revealed by RAPD markers

Although Azerbaijan is a center of natural distribution and diversity of genus Quercus around the world, less published data can be found about PCR-based genetic diversity in prevalent oak accessions. The present study aims at finding molecular variation among the Quercus species using RAPD markers. In this study, genomic fingerprinting in five Azerbaijan oak species (Quercus spp.) was carried out using 10 RAPD markers. The result showed that a total of 113 DNA fragments were amplified, of those, 91 bands were polymorphic. The highest PIC, EMR and MI values was observed in the primers OPD-05, OPB-01 and OPB-03. The genetic similarity among the genotypes examined ranged from 0.333 to 0.818 with an average of 0.498. Species-specific DNA fragments were found in all species examined. Based on the cluster analysis using UPGMA method and Jaccard’s similarity index, all the examined oak species fell into three main groups and principal coordinate analysis (PCoA) supported the clustering result. High genetic diversity was found in the tested genetic germplasm of oak species. The results of this study would be very useful for oak improvement program.

Antibacterial activity of crude protein and development of species-specific molecular marker for fungus Ophiocordyceps sobolifera

The medicinal mushroom Ophiocordyceps sobolifera has been used in traditional Chinese medicine; however, the proven medical benefits of the crude protein are limited. Therefore, in this study, forty-two isolates of O. sobolifera were identified using their internal transcribed spacer (ITS) sequences. Then, the antibacterial activities of the crude proteins were evaluated against strains of methicillin-resistant and -susceptible Staphylococcus aureus and Bacillus cereus using agar well diffusion technique. The results revealed that the crude protein had a good inhibitory effect against S. aureus and B. cereus. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of the crude proteins ranged from 0.156–10.00 mg/mL and 0.156–10.00 mg/mL, respectively. Among them, the Cod-NB1302 strain had the broadest inhibitory activity against the tested bacterial pathogens with MIC values of 0.625–1.25 mg/mL and MBC values of 0.625–1.25 mg/mL. Then, the species-specific molecular marker derived from random amplified polymorphic DNA (RAPD) was developed for the identification of O. sobolifera. The results indicated that the OPT13 arbitrary primer was able to generate two species-specific DNA fragments from O. sobolifera. Specific fragments were then cloned, sequenced, and used to design the species-specific primers. Only one species-specific primer set, the SpsoL_f01/SpsoL_r01 primer pair, produced a prominent amplicon of 118 bps in O. sobolifera under stringent conditions without non-specific detection after being applied to other species, with the exception of O. longissima. Moreover, the detection limit of this primer was 5.84 ng of genomic DNA, and this primer set could be applied in all strains of O. sobolifera.

Population Genetic Relationships between Mouflon and Domesticated Sheep Breeds in Highly Polymorphic Genomic Elements

A comparative analysis of population-genetic structures of three indigenous sheep breeds (Kalmyk, Edilbay, and Karachay breeds) and closely related wild species of the European Mouflon has been performed with polylocus genotyping at the highly polymorphic fragments of genomic DNA flanked by inverted repeats of microsatellite loci and a long terminal repeat region of endogenous retrovirus BERV-K1 (ISSR-PCR and IRAP-PCR markers). Increased genetic heterogeneity of ISSR-PCR and IRAP-PCR markers in males compared to females is revealed in all of the tested animal groups. The spectra of amplification products, including breed- and species-specific genomic DNA fragments, are identified. Genotyping for them may contribute to the control over the process of consolidation of breeds and intrabreed groups. Representatives of the European Mouflon are found to be different from the domesticated sheep in higher-rank polymorphism in the fragments of genomic DNA flanked by inverted repeats of BERV-K1, which may indicate the mobile genetic elements involved in the population-genetic differentiation between the domesticated and closely related wild species.

Assessment of dual schistosome infection prevalence from urine in an endemic community of Ghana by molecular diagnostic approach

Schistosomiasis is an important Neglected Tropical Disease caused by blood parasites called schistosomes. In sub-Saharan Africa, two major human schistosomes, namely Schistosoma mansoni and S. haematobium, often occur sympatrically and is responsible for almost 90% of the affected 290 million people worldwide. We have utilized a highly sensitive and specific assay by amplifying species-specific cell-free repeat DNA fragments by polymerase chain reaction to detect either single or dual schistosome infection from a single urine sample from a broad age group. In this study, we have tested filtered urine samples collected from 163 individuals aged 3–63 years, mostly children (median age 10), to evaluate the prevalence of single and dual infections for S. mansoni and S. haematobium in Tomefa community in the Greater Accra region of Ghana. 40–50 mL of urine was filtered through a 12.5 cm Whatman # 3 filter paper in the field. The filter papers were dried, packed individually in sealable plastic bags with a desiccant, and shipped to Marquette University, where DNA was isolated and PCR amplification was carried out with species-specific primers. Disease prevalence was found to be 46.6% for S. mansoni and 48.5% for S. haematobium. Most importantly, 23.3% of participants had dual infections. All of the samples were detected without any cross amplification. The data was evaluated for four age groups and infection rate was highest for the age group of 3–12 years, with more S. haematobium infections than S. mansoni infections. We found a high prevalence of both S. haematobium and S. mansoni infection and a significant proportion of dual infection for the Tomefa community, which in most cases would be missed by traditional parasitological examination of urine or stool. Our highly sensitive and specific approach for detecting underlying multiple schistosome infections is an effective means to detect low intensity infections and would enhance the effectiveness of surveillance and Mass Drug Administration control programs of schistosomiasis.

Detection of elusive fire salamander larvae (Salamandra salamandra) in streams via environmental DNA

Abstract In the face of the global biodiversity crisis, the monitoring of species richness and diversity is experiencing an increased demand entailing a raise in cost and time investment. The analysis of species-specific DNA fragments in environmental samples (eDNA) such as from water or soil, facilitate the molecular detection of species without the specific sampling of individuals. The invasive chytrid fungus Batrachochytrium salamandrivorans (Bsal) is infecting natural fire salamander populations (Salamandra salamandra) and causes chytridiomycosis resulting in infrequent regional extinctions of populations across Central Europe. With regard to the expanding distribution of Bsal over the last years, cost-effective monitoring of fire salamanders is important for the conservation of this species. Based on a real-time quantitative PCR (qPCR) assay, we developed a new protocol to detect S. salamandra larvae in streams via eDNA, using species-specific primers of the mitochondrial control region (D-loop). We tested the efficiency of qPCR primer sets for six combinations of DNA extraction kits coupled with subsequent PCR inhibitor removal kits for obtaining qPCR-detectable S. salamandra eDNA from water filters, that were taken both from natural streams and artificial water tanks in the laboratory as positive controls. We found that the DNeasy Blood & Tissue Kit in combination with the DNeasy PowerClean CleanUp Kit performed best for detecting salamander larvae from natural streams. Our experimental protocol paves the way for resource-saving approaches to monitor S. salamandra larvae, but also confirms the limits to this eDNA approach in that it requires optimized laboratory protocols.

Molecular genetic identification of arbuscular mycorrhizal fungi

Arbuscular mycorrhiza (AM) is a widespread symbiosis formed by most land plants with fungi from Glomeromycotina subdivision. The main problem in study of AM fungi is the complication in identification, associated with high intra- and interspecific genetic polymorphism, as well as obligate status of AM fungi in relation to host plant. The methodology for AM fungi identification is constantly undergoing major changes. In the review the selection of optimal methods of molecular genetic identification for AM fungi is carried out. The sample preparation, selection of species-specific marker DNA fragments and primers design, amplification including nested PCR are considered. The prospects for cloning and next generation sequencing for AM fungi identification are analyzed and substantiated.

Development of SCoT-Based SCAR Marker for Rapid Authentication of Taxus Media.

Taxus media is an important species in the family Taxaceae with high medicinal and commercial value. Overexploitation and illegal trade have led T. media to a severe threat of extinction. In addition, T. media and other Taxus species have similar morphological traits and are easily misidentified, particularly during the seedling stage. The purpose of this study is to develop a species-specific marker for T. media. Through a screening of 36 start codon targeted (SCoT) polymorphism primers, among 15 individuals of 4 Taxus species (T. media, T. chinensis, T. cuspidate and T. fuana), a clear species-specific DNA fragment (amplified by primer SCoT3) for T. media was identified. After isolation and sequencing, a DNA sequence with 530bp was obtained. Based on this DNA fragment, a primer pair for the sequence-characterized amplified region marker was designed and named MHSF/MHSR. PCR analysis with primer pair MHSF/MHSR revealed a clear amplified band for all individuals of T. media but not for T. chinensis, T. cuspidate and T. fuana. Therefore, this marker can be used as a quick, efficient and reliable tool to identify T. media among other related Taxus species. The results of this study will lay an important foundation for the protection and management of T. media as a natural resource.

Detection of duo-schistosome infection from filtered urine samples from school children in Zambia after MDA.

Schistosomiasis is one of the major Neglected Tropical Diseases (NTDs) in sub-Saharan Africa. In sub-Saharan Africa, two major human schistosome species namely Schistosoma mansoni and S. haematobium often occur sympatrically largely affecting children. Recognizing the public health impact of Schistosomiasis, the World Health Organization (WHO) is urging member states to regularly treat at least 75% and up to 100%, of all school-aged children at risk of morbidity. For control strategies based on targeted mass drug administration (MDA) to succeed it is essential to have a simple and sensitive test for monitoring the success of these interventions. Current available diagnostic tests, such as egg detection in stool by Kato-Katz (KK) for S. mansoni and detection of eggs or blood (hematuria) in urine for S. haematobium have reduced sensitivity in low intensity settings. The objective of the study was to evaluate active single or duo schistosome infections in school children following MDA using molecular diagnostics (PCR) on filtered urine samples and comparing that against traditional diagnostic tests. This cross-sectional study was conducted among 111 school children aged 7–15 years in Chongwe and Siavonga Districts in Zambia. Species-specific cell-free repeat DNA fragment were amplified from 111 filtered urine samples. Our approach detected eight times more positive cases (total 77) than by KK (9) for S. mansoni and six times more (total 72) than by hematuria (11) for S. haematobium and even more against urine filtration (77 compared to only 6). The same pattern was observed when stratified for age group and sex specific analysis with 100% sensitivity and specificity devoid of any cross amplification. In addition, 69 individuals (62%) were co-infected by both parasites. We have demonstrated a significantly higher prevalence of both species than indicated by the traditional tests and the persistent maintenance of reservoir of infection after MDA. Our approach is an effective means of detecting low intensity infection, which will enhance the effectiveness of surveillance and assess the impact of MDA control programs against schistosomiasis.

Point of care diagnosis of multiple schistosome parasites: Species-specific DNA detection in urine by loop-mediated isothermal amplification (LAMP)

Schistosomes are easily transmitted and high chance of repeat infection, so if control strategies based on targeted mass drug administration (MDA) are to succeed it is essential to have a test that is sensitive, accurate and simple to use. It is known and regularly demonstrated that praziquantel does not always eliminate an infection so in spite of the successes of control programs a residual of the reservoir survives to re-infect snails. The issue of diagnostic sensitivity becomes more critical in the assessment of program effectiveness. While serology, such as antigen capture tests might improve sensitivity, it has been shown that the presence of species-specific DNA fragments will indicate, most effectively, the presence of active parasites. Polymerase chain reaction (PCR) can amplify and detect DNA from urine residue captured on Whatman No. 3 filter paper that is dried after filtration. Previously we have detected S. mansoni and S. haematobium parasite-specific small repeat DNA fragment from filtered urine on filter paper by PCR. In the current study, we assessed the efficacy of detection of 86 urine samples for either or both schistosome parasites by PCR and loop-mediated isothermal amplification (LAMP) that were collected from a low to moderate transmission area in Ghana. Two different DNA extraction methods, standard extraction kit and field usable LAMP-PURE kit were also evaluated by PCR and LAMP amplification. With S. haematobium LAMP amplification for both extractions showed similar sensitivity and specificity when compared with PCR amplification (100%) verified by gel electrophoresis. For S. mansoni sensitivity was highest for LAMP amplification (100%) for standard extraction than PCR and LAMP with LAMP-PURE (99% and 94%). The LAMP-PURE extraction produced false negatives, which require further investigation for this field usable extraction kit. Overall high positive and negative predictive values (90% − 100%) for both species demonstrated a highly robust approach. The LAMP approach is close to point of care use and equally sensitive and specific to detection of species-specific DNA by PCR. LAMP can be an effective means to detect low intensity infection due to its simplicity and minimal DNA extraction requirement. This will enhance the effectiveness of surveillance and MDA control programs of schistosomiasis.

Naturally occurring triploid hybrids between Miscanthus sacchariflorus and M. sinensis in Southern Japan, show phenotypic variation in agronomic and morphological traits

Miscanthus × giganteus, a triploid hybrid between tetraploid M. sacchariflorus and diploid M. sinensis, is considered to be a promising biomass crop for producing renewable bioenergy in temperate regions. However, because most cultivars of the hybrid have been derived from a single clone through vegetative propagation, genetic resources with diverse phenotypes are required to allow adaptation to changing environments and resistance to pests and diseases. The present study aimed to identify novel natural hybrids between M. sacchariflorus and M. sinensis and to assess their natural variation in agronomic and morphological traits. We have identified 51 Miscanthus plants with nuclear DNA content of triploid Miscanthus within a 5-km radius of the Kikuchigawa River basin located in southern Japan. Seven spontaneous triploids, including five representative strains in the Kikuchigawa River basin, had the chromosome number and nuclear DNA content comparable with those of triploid M. × giganteus, and showed the amplification of species-specific DNA fragments from both parent species in DNA marker analysis. These demonstrated that these spontaneous strains were natural triploid hybrids. A 2-year-long field experiment in a warm climate revealed that these triploid hybrids showed phenotypic variation in agronomic and morphological traits. In addition, phylogenetic analysis of chloroplast DNA sequences revealed that these triploid hybrids had a polyphyletic origin but that all seven hybrids contained cytoplasm from M. sacchariflorus. The present study suggests that naturally occurring triploid hybrids could be more widely distributed in southern Japan than previously reported and be important breeding resources to develop Miscanthus cultivars with novel characteristic traits.

Development of molecular markers for authentication of the medicinal plant species Patrinia by random amplified polymorphic DNA (RAPD) analysis and multiplex-PCR

The authentication of components from plant species is vital for the quality control and standardization of herbal medicines. Due to morphological similarities when dried, it is very difficult to distinguish authentic medicinal plants Patrinia villosa and P. scabiosifolia from adulterants of P. rupestris and P. saniculifolia in the herbal medicine Patriniae Radix. To establish a reliable authentication tool distinguishing authentic Patriniae Radix species from adulterants, we used random amplified polymorphic DNA (RAPD) genomic profiling and obtained several species-specific DNA fragments for each of the four Patrinia species. Based on the sequences of these amplicons, we developed sequence characterized amplified region (SCAR) markers as stable molecular authentication tools. SCAR markers to identify the authentic Patriniae Radix species, P. villosa and P. scabiosifolia, were developed. To accurately identify adulterants, we also developed SCAR markers able to identify the inauthentic P. rupestris and P. saniculifolia species. Furthermore, we established multiplex-PCR SCAR assays to simultaneously distinguish all four species using combinations of species-specific SCAR marker primer sets. These efforts resulted in reliable DNA-based molecular markers to identify authentic Patriniae Radix species from their adulterants and a rapid authentication assay for the efficient identification of Patrinia species. These markers will facilitate the standardization of Patriniae Radix preparations and therefore prevent the distribution of adulterants.

Comparative Estimate of the sheep breed gene pools using ISSR-analysis

Using the ISSR-PCR technique, the genetic structure of nine sheep breeds (Ovis aries) bred on the territories of Russia and Mongolia was examined. Species-specific and breed-specific DNA fragments were identified. For the first time, data on the genetic diversity of Telengit and Buubey sheep breeds were obtained. The main parameters of the genetic diversity and the breed structure were assessed, and the phylogenetic relationships and genetic distances between the studied breeds were determined. Using the method of hierarchical frequency averaging, the prototypal sheep gene pool was reconstructed. The three-tiered analysis of diversity based on the ISSR fingerprinting data showed that 15.8% of variability was found between the breeds, 31.4% of variability was found between the populations within the breeds, and the diversity among the individuals within the populations constituted 52.8%.

Accurate diagnostics for schistosomiasis: a new role for PCR?

Accurate diagnostics for schistosomiasis: a new role for PCR? Clive Shiff Harry W Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA Abstract: When it is important to detect schistosome infection with high sensitivity, particularly where local elimination is planned, detection of parasite-specific DNA should be considered as part of the diagnosis. The process of molecular amplification need no longer be considered to be restricted to sophisticated laboratories, but can be used effectively with modern tools in district hospitals or rural clinics and can be adapted for urine if it is used as the DNA source. With schistosomiasis, observation of a typical egg in urine, stool, or biopsy is taken as evidence of infection; point-of-care (POC) tests based on detecting parasite-specific antigens on a simple matrix have made this simpler and effective particularly in rural settings, but like observation of eggs, these methods are liable to give false negative results and thus miss infected cases. These cases are a likely source of parasite eggs to continue the infection cycle. Thus, although convenient, POC tests are unsatisfactory for epidemiological studies and evaluation of mass treatment campaigns. Methods to detect species-specific DNA fragments in fresh urine or in residue after filtration have been described and shown to be more sensitive and specific than specimen-based, serological, or antigen capture diagnosis. Parasite-specific DNA fragments can also be amplified from blood and stool taken from people infected with any of the major schistosomes. However, the use of urine residue after filtering through filter paper as a source of parasite-specific DNA has greatly simplified the process. Urine can be filtered at the POC using disposable plasticware and then paper dried and placed in a plastic sleeve, which is a simple process. If dry, the product is stable and transport is simple and inexpensive, and DNA can be extracted and amplified in a nearby facility set up for the work. If not POC, this is close to POC and quite feasible and processing by PCR or loop-mediated isothermal amplification process will ensure the universal application of this technology. Keywords: schistosome differential diagnosis, urine-based test, DNA fragments, PCR, LAMP&nbsp

Application of Quadruple Multiplex PCR Detection for Beef, Duck, Mutton and Pork in Mixed Meat

In this study, we have developed quadruple multiplex PCR assay for meat (beef, duck, mutton and pork) in processed meat products. By mixing four primers in appropriate ratios could be identified by the PCR. A forward primer was designed on a conserved DNA sequence in the mitochondrial ND2 and 16S rDNA gene in sheep and duck genes, and reverse primers on species-specific DNA sequences for each species. PCR primers were designed to give different length fragments from the four meats. The products showed species-specific DNA fragments of 116, 212, 177 and 322 bp from beef, pork, mutton and duck. Optimal PCR conditions were established. The assay sensitivity under these conditions was 0.1ng, and its specificity was 100 %. The results of the study suggest that PCR represents a simple, efficient test method as a practical alternative for the rapid detection and identification of meat.

Rapid identification of goat (Capra hircus) and sheep (Ovis aries) species in raw meat using duplex PCR assay

Adulteration of high quality meat with their inferior/cheaper counterparts has become a common practice in the meat industry, which is not detectable by the naked eye or by eating. To circumvent this problem, molecular methods had been developed. The present study was carried out for detection of meat species with the use of cytochrome b gene variability by duplex PCR. Meat samples from goat, sheep and buffalo were utilized for molecular analysis. Genomic DNA was isolated from six samples of each species with some modifications. Mitochondrial cyotchrome b gene was amplified by conventional and duplex PCR using a common forward primer and species-specific reverse primer. PCR amplicons were resolved by agarose gel electrophoresis and for each species produce a characteristic band pattern in conventional and duplex PCR was obtained. The PCR products showed species-specific DNA fragments of 157 and 331 bps from goat and sheep respectively. The duplex PCR could detect upto 10 % of DNA of meat species. Thus, the duplex PCR was found to be a simple, reliable, sensitive and highly specific test for simultaneous detection of meat species.