Species-specific Polymerase Chain Reaction Assay Research Articles

Species-specific loop-mediated isothermal amplification (LAMP) assay for identification of tissue of cattle origin by targeting mitochondrial gene sequences.

The present study was carried out with the objective of development of species-specific loop-mediated isothermal amplification (LAMP) assay for identification of tissue of cattle origin. The cattle-specific LAMP primer set was designed by targeting mitochondrial D-loop gene. The conditions for LAMP reaction for amplification of template DNA from cattle using designed cattle-specific primer set were optimized for the components of mixture and temperature of reaction. Amplified products were analysed using SYBR Green I dye and by agarose gel electrophoresis. The developed species-specific LAMP assay was evaluated for its specificity, sensitivity and validated in laboratory on samples from known, coded, binary meat admixture with other than cattle at relative percentage of 20%, 10%, 5% and 1%, Phire tissue direct PCR master mix treated tissues of cattle and on species-specific polymerase chain reaction assay positive samples. The developed LAMP assay using self-designed primer set was highly specific, amplifying the DNA template exclusively from cattle tissue under the optimized LAMP reaction conditions. The sensitivity assay using serially diluted DNA templates revealed lowest level of detection as 0.01ng of absolute DNA from target species. Laboratory validation substantiated the accuracy of assay in known/unknown (coded) samples and up to the 1% level of admixture in binary meat sample. DNA present in supernatant of Phire Animal tissue kit treated samples were also amplified successfully eliminating the extra step of extraction of genomic DNA. The developed assays exhibited comparable results with previously established species-specific PCR assay taken as gold standards. Thus, it was concluded that developed species-specific loop-mediated isothermal amplification assay was effective in identification of tissue of cattle origin.

Mislabeling in Cooked Sausage is a Seriously Increasingly Problem in Food Safety

Background: Identifying the animal species origin in meat and meat products is important for preventing adulteration and protecting consumers in terms of health and religious convictions. Species-specific polymerase chain reaction (PCR) is known as a suitable method for identifying meat species. OBJECTIVES: This study aimed to use a species-specific PCR assay for the detection of mislabeling in cooked sausage meats as adulterants by use of multiplex PCR. METHODS: A total of 114 samples including sausage labeled containing 40%, 55% and 70% red meat of 10 different brands were collected from various markets and supermarkets. Following genomic DNA extraction from cooked sausages which were claimed to be made of red meat, multiplex PCR was performed to detect adulteration in processed food. RESULTS: According to the analysis, 60 sausage samples showed that they consist of only meat from chicken (52.6%), 48 sausage samples consist of meat from beef and chicken (42.1%) and only 5.3% of the examined sausages were prepared with the meat of beef (6 samples). CONCLUSIONS: This high rate of undeclared chicken meat in sausage samples is most probably due to achieving more profit. Our results indicated that the meat species substitution occurs often in processed meats like sausages, which indicates the need of more governmental controls.

Improving cost-efficiency of faecal genotyping: New tools for elephant species.

Despite the critical need for non-invasive tools to improve monitoring of wildlife populations, especially for endangered and elusive species, faecal genetic sampling has not been adopted as regular practice, largely because of the associated technical challenges and cost. Substantial work needs to be undertaken to refine sample collection and preparation methods in order to improve sample set quality and provide cost-efficient tools that can effectively support wildlife management. In this study, we collected an extensive set of forest elephant (Loxodonta cyclotis) faecal samples throughout Gabon, Central Africa, and prepared them for genotyping using 107 single-nucleotide polymorphism assays. We developed a new quantitative polymerase chain reaction (PCR) assay targeting a 130-bp nuclear DNA fragment and demonstrated its suitability for degraded samples in all three elephant species. Using this assay to compare the efficacy of two sampling methods for faecal DNA recovery, we found that sampling the whole surface of a dung pile with a swab stored in a small tube of lysis buffer was a convenient method producing high extraction success and DNA yield. We modelled the influence of faecal quality and storage time on DNA concentration in order to provide recommendations for optimized collection and storage. The maximum storage time to ensure 75% success was two months for samples collected within 24 hours after defecation and extended to four months for samples collected within one hour. Lastly, the real-time quantitative PCR assay allowed us to predict genotyping success and pre-screen DNA samples, thus further increasing the cost-efficiency of our approach. We recommend combining the validation of an efficient sampling method, the build of in-country DNA extraction capacity for reduced storage time and the development of species-specific quantitative PCR assays in order to increase the cost-efficiency of routine non-invasive DNA analyses and expand the use of next-generation markers to non-invasive samples.

It’s Gut Check Time! A Universal Food Immunomarking Technique for Studying Arthropod Feeding Activities

The analysis of arthropod feeding activity is often determined by using species-specific postmortem gut content polymerase chain reaction and enzyme-linked immunosorbent assays (ELISA). Such mono-specific assays require time, resources, and technical expertise to develop for the food item (usually a pest insect species) that is the target of the investigation. A generic predator gut analysis method was described over a quarter of a century ago that does not require the development of a species-specific gut assay. This generic method remained in relative obscurity until about a decade ago. Recently, it has been used to study a wide range of arthropod feeding activities, such as carnivory, herbivory, scavenging, and other feeding interactions. For this review, I have coined this method as the universal food immunomarking technique (UFIT). The UFIT consists of tagging food items (i.e., prey, foliage, carrion, etc.) with a specific protein. In turn, the gut contents of foraging arthropods are examined for the presence of protein-marked food items by a standardized protein-specific sandwich ELISA. In this article, I give examples of the benefits of the UFIT gut assay approach over prey-specific gut assay approaches and tips on conducting a successful UFIT experiment, and provide examples of how it has been adapted to study a wide variety of arthropod feeding behaviors. My goal is to make researchers aware of another valuable tool in the gut analysis toolbox.

Screening for Exotic Forest Pathogens to Increase Survey Capacity Using Metagenomics.

Anthropogenic activities have a major impact on the global environment. Canada's natural resources are threatened by the spread of fungal pathogens, which is facilitated by agricultural practices and international trade. Fungi are introduced to new environments and sometimes become established, in which case they can cause disease outbreaks resulting in extensive forest decline. Here, we describe how a nationwide sample collection strategy coupled to next-generation sequencing (NGS) (i.e., metagenomics) can achieve fast and comprehensive screening for exotic invasive species. This methodology can help provide guidance to phytopathology stakeholders such as regulatory agencies. Several regulated invasive species were monitored by processing field samples collected over 3 years (2013 to 2015) near high-risk areas across Canada. Fifteen sequencing runs were required on the Ion Torrent platform to process 398 samples that yielded 45 million reads. High-throughput screening of fungal and oomycete operational taxonomic units using customized fungi-specific ribosomal internal transcribed spacer 1 barcoded primers was performed. Likewise, Phytophthora-specific barcoded primers were used to amplify the adenosine triphosphate synthase subunit 9-nicotinamide adenine dinucleotide dehydrogenase subunit 9 spacer. Several Phytophthora spp. were detected by NGS and confirmed by species-specific quantitative polymerase chain reaction (qPCR) assays. The target species Heterobasidion annosum sensu stricto could be detected only through metagenomics. We demonstrated that screening target species using a variety of sampling techniques and NGS-the results of which were validated by qPCR-has the potential to increase survey capacity and detection sensitivity, reduce hands-on time and costs, and assist regulatory agencies to identify ports of entry. Considering that early detection and prevention are the keys in mitigating invasive species damage, our method represents a substantial asset in plant pathology management.

Molecular Characterization and Identification of Stubby Root Nematode Species From Multiple States in the United States.

Stubby root nematodes (SRN) are important plant parasites infecting many crops and widely distributed in many regions of the United States. SRN transmit Tobacco rattle virus, which causes potato corky ringspot disease, thereby having a significant economic impact on the potato industry. In 2015 to 2017, 184 soil samples and 16 nematode suspensions from North Dakota, Minnesota, Idaho, Oregon, Washington, South Carolina, North Carolina, and Florida were assayed for the presence of SRN. SRN were found in 106 soil samples with population densities of 10 to 320 SRN per 200 g of soil and in eight of the nematode suspensions. Sequencing of ribosomal DNA (rDNA) or species-specific polymerase chain reaction assays revealed the presence of four SRN species, including Paratrichodorus allius, P. minor, P. porosus, and Trichodorus obtusus. Accordingly, their rDNA sequences were characterized by analyzing D2-D3 of 28S rDNA, 18S rDNA, and internal transcribed spacer (ITS) rDNA obtained in this study and retrieved from GenBank. Both intra- and interspecies variations were higher in ITS rDNA than 18S rDNA and D2-D3 of 28S rDNA. Based on phylogenetic analysis, the four SRN species formed a monophyletic group, with P. allius more closely related to P. porosus than P. minor and T. obtusus. Indel variation of ITS2 rDNA was present in P. allius populations from the same geographic regions. This study documented the occurrence of SRN species across multiple states. The intra- and interspecies genetic diversity of rDNA in this study will provide more information for understanding the evolutionary relationships of SRN and will be valuable for future studies of SRN species identification and management.

First Record of the Invasive Pest Pseudococcus jackbeardsleyi (Hemiptera: Pseudococcidae) on the Chinese Mainland and Its Rapid Identification Based on Species-Specific Polymerase Chain Reaction.

The Jack Beardsley mealybug, Pseudococcus jackbeardsleyi Gimpel & Miller (Hemiptera: Pseudococcidae), a globally devastating, invasive polyphagous insect, causes great damage to many fruits, ornamentals, vegetables, and food crops worldwide. It is of Neotropical origin and has invaded throughout America and in parts of Asia, Africa and Oceania, and is still expanding its invasion ranges. Therefore, a method for quick and correct identification of this invasive species is crucial for quarantine and spreading interruption of it. In present study, we report the first record of P. jackbeardsleyi on the Chinese mainland, which would cause great damage to many crops. The identification of P. jackbeardsleyi was verified via morphological characters and DNA barcoding. One pair of species-specific polymerase chain reaction (SS-PCR) primers was designed based on variations in the sequences of the mitochondrial cytochromecoxidasesubunitI gene among P. jackbeardsleyi and 28 other mealybug species. No cross-reaction was detected among 21 closely related species using this SS-PCR assay, demonstrating the specificity of this marker. Furthermore, this method was successfully applied to detect individuals from different developmental stages and adult debris across four geographic populations of P. jackbeardsleyi, showing the high stability of the assay. Additionally, the detection limit of the marker was 55.94 ± 5.05 pg/µl of P. jackbeardsleyi DNA, illustrating the high sensitivity of the assay. The SS-PCR assay developed in this study provides a rapid, simple and reliable method for the identification of P. jackbeardsleyi, which should be crucial in the plant quarantine, early detection and sustainable management of this globally invasive pest.

Serum based polymerase chain reaction and enzyme linked immunosorbent assays for diagnosis of bovine brucellosis

Brucellosis is one of the economically important diseases in India and diagnosis of brucellosis using single test is cumbersome due to variation in sensitivity and specificity among the different test. The present study was aimed to assess the suitability of serum as clinical specimen in molecular diagnosis and evaluate the serology and molecular assays as in diagnosis of bovine brucellosis. A total of 821 bovine sera samples were subjected to indirect Enzyme Linked Immunosorbent Assay (i-ELISA) and serum based Polymerase Chain Reaction (PCR) assay. On serology 6.70 per cent positivity of brucellosis were reported and on PCR assay, 47 and 29 sera samples were positive for bcsp 31 genus specific and IS711 species specific PCR assay respectively with per cent positivity of 5.72 and 3.53. In comparison between serology and molecular test, 44 samples were positive for both assays and 11 and 3 samples were positive for serology and molecular assays individually. This study suggests that serum sample can be utilised as the choice of clinical specimen for both PCR assay and i-ELISA will be a future choice for the diagnosis of bovine brucellosis.

Monitoring of Red Pepper Powder and Seasoned Red-Pepper Sauce using Species-Specific PCR in Conjunction with Whole Genome Amplification

Red pepper is one of the most important spices popularly utilized in Korea. Because of the differences in tariff rates between red pepper powder and seasoned red-pepper sauce, seasoned red-pepper sauce is often therefore imported by consumers, then dried, ground, and added to red pepper powder for cost effective purposed to use the product the most effectively. In this study, we combined species-specific polymerase chain reaction (PCR) assays (for red pepper, garlic, onion, spring onion, and ginger) with whole-genome amplification (WGA). Thirty-nine red pepper powders were well in accordance with their labels. However, six red pepper powder and five seasoned redpepper sauce products failed to meet their compliance requirements. As a consequence, our monitoring results revealed that the overall mislabeling rate detected in this study was identified at 22%. Thus, our findings showed that the species-specific PCR in conjunction with WGA was an ideal method to identify raw materials that are used in the manufacturing of red pepper powder and seasoned red-pepper sauce.

Molecular Detection of Chevon and Mutton Adulteration by Species-specific PCR Assay Targeting Mitochondrial COI Gene

A highly species-specific polymerase chain reaction (PCR) was developed for authentic identification of raw and processed meat products of chevon and mutton. To achieve this, four species-specific primers for mitochondrial cytochrome oxidase I (COI) gene were selected from previous reports. The assay generated PCR products of 157, 157, 268, 251, and 177 bp for chevon, mutton, pork, chicken and duck, respectively. The sensitivity for the detection of adulteration was established to be 0.1% (w/w), while the DNA limit for detection was as low as 0.001 ng. The adulteration was found in all meat product types including raw frozen meat, cold cuts, ground meats and cooked foods. These findings showed that species-specific PCR are potentially reliable method in detection of meat products of chevon and mutton authentication.

Molecular characterization of Dirofilaria spp. circulating in Portugal

BackgroundDirofilariosis is a potentially zoonotic parasitic disease, mainly transmitted by mosquito vectors in many parts of the world. Data concerning the canine Dirofilaria species currently circulating in Portugal is scarce. Thereby, a large-scale study was conducted to determine the Dirofilaria spp. present in Portugal, based on a molecular approach, and also to optimize a reliable and highly sensitive species-specific polymerase chain reaction (PCR) assay that could be used for the simultaneous detection and differentiation of Dirofilaria immitis, Dirofilaria repens, and other concurrent filarial species in animal reservoirs.MethodsBlood samples were collected from three districts of Portugal (Coimbra, Santarém and Setúbal) between 2011 and 2013. Samples were tested using rapid immunomigration tests (Witness® Dirofilaria), modified Knott’s technique and acid phosphatase histochemical staining. In addition, molecular analysis was performed by amplification of the internal transcribed spacer (ITS) region using two different PCR protocols, specific for molecular screening of canine filarial species.ResultsOf the 878 dogs sampled, 8.8% (n = 77) were positive for D. immitis circulating antigen and 13.1% (n = 115) positive for microfilariae by the modified Knott’s technique. Of the 134 samples tested by acid phosphatase histochemical staining, 100 (74.6%) were positive for D. immitis. Overall, 13.7% (n = 120) were positive by PCR for D. immitis by ITS2, of which 9.3% (67/720) were also positive by ITS1. ITS2 PCR was the most sensitive and specific method, capable of detecting mixed D. immitis and A. reconditum infections. Heterozygosity, in the form of double peaks, was detected by sequencing of both ITS regions. No D. repens was detected by any of the diagnostic methods.ConclusionsThe present study confirmed D. immitis as the dominant species of the genus Dirofilaria infecting Portuguese dogs, based on sequencing of ITS1 and ITS2 PCR fragments. Additionally, ITS2 PCR was the most adequate method for diagnosis and prevalence estimation.

Simultaneous detection of bovine and porcine DNA in pharmaceutical gelatin capsules by duplex PCR assay for Halal authentication

BackgroundIn the pharmaceutical industry, hard- and soft-shelled capsules are typically made from gelatin, commonly derived from bovine and porcine sources. To ensure that pharmaceutical products comply with halal regulations in Muslim countries (no porcine products allowed), development of a valid, reliable, quick, and most importantly, cost-effective tests are of utmost importance.MethodsWe developed a species-specific duplex polymerase chain reaction (PCR) assay targeting 149 bp porcine and 271 bp bovine mitochondrial DNA (mtDNA) to simultaneously detect both porcine and bovine DNA (in one reaction at the same time) in gelatin. Some additional simplex PCR tests (targeting 126 bp bovine and 212 bp porcine mtDNA) and real-time PCR using a commercially available kit (for identification of porcine DNA) were used to verify the selectivity and sensitivity of our duplex PCR. After optimization of DNA extraction and PCR methods, hard/soft pharmaceutical gelatin capsules (containing drug) were tested for the presence of porcine and/or bovine DNA.ResultsDuplex PCR detected the presence of as little as 0.1% porcine DNA, which was more accurate than the commercially available kit. Of all gelatin capsules tested (n = 24), 50% contained porcine DNA (pure porcine gelatin alone or in combination with bovine gelatin).ConclusionsDuplex PCR presents an easy-to-follow, quick, low-cost and reliable method to simultaneously detect porcine and bovine DNAs (>100 bp) in minute amounts in highly processed gelatin-containing pharmaceutical products (with a 0.1% sensitivity for porcine DNA) which may be used for halal authentication.Graphical abstractSimultaneous detection of porcine and bovine DNA in gelatin capsules by duplex PCR

Lytic Activity of Various Phage Cocktails on Multidrug-Resistant Bacteria

A bacteriophage is a virus that infects and replicates within a bacterium following the injection of phage genome into the bacterial cytoplasm. They are seen as a possible therapy for multi-drug-resistant strains of many bacteria. The aim of this study is to evaluate the lytic activity of the Pyo, Intesti and Fersisi bacteriophage cocktails on P. aeruginosa and S. aureus. Ten different S. aureus and P. aeruginosa strains, which were isolated from hospitalized patients in Turkey, were used in the study. The identification and antibiotic susceptibility of the isolates were performed using Vitec 2 system. The identities of the isolates were confirmed by a species-specific Polymerase Chain Reaction (PCR) assay. Lytic activity of the bacteriophage cocktails on bacteria was determined by spot test and plaque assay methods. The lytic activity of the Pyo phage cocktail was evaluated on P. aeruginosa and S. aureus strains. It was found that eight isolates of MDR S. aureus were susceptible to Pyo phage cocktail and two isolates were resistant. Nine isolates of antibiotic-resistant P. aeruginosa were found to be susceptible to this phage cocktail and one isolate was resistant. Thus, the Pyo, Intesti and Fersisi cocktails are very effective in treating clinical strains of multidrug-resistant P. aeruginosa and S. aureus isolated in Turkey. The Pyo, Intesti and Fersisi cocktails may prove useful in the treatment of various infections caused by those bacteria.

Spatiotemporal Patterns in the Airborne Dispersal of Spinach Downy Mildew.

Downy mildew is the most devastating disease threatening sustainable spinach production, particularly in the organic sector. The disease is caused by the biotrophic oomycete pathogen Peronospora effusa, and the disease results in yellow lesions that render the crop unmarketable. In this study, the levels of DNA from airborne spores of P. effusa were assessed near a field of susceptible plants in Salinas, CA during the winter months of 2013-14 and 2014/15 using rotating-arm impaction spore-trap samplers that were assessed with a species-specific quantitative polymerase chain reaction (qPCR) assay. Low levels of P. effusa DNA were detectable from December through February in both winters but increased during January in both years, in correlation with observed disease incidence; sharp peaks in P. effusa DNA detection were associated with the onset of disease incidence. The incidence of downy mildew in the susceptible field displayed logistic-like dynamics but with considerable interseason variation. Analysis of the area under the disease progress curves suggested that the 2013-14 epidemic was significantly more severe than the 2014-15 epidemic. Spatial analyses indicated that disease incidence was dependent within an average range of 5.6 m, approximately equivalent to the width of three planted beds in a typical production field. The spatial distribution of spores captured during an active epidemic most closely fit a power-law distribution but could also be fit with an exponential distribution. These studies revealed two important results in the epidemiology of spinach downy mildew in California. First, they demonstrated the potential of impaction spore-trap samplers linked with a qPCR assay for indicating periods of high disease risk, as well as the detection of long-distance dispersal of P. effusa spores. Second, at the scale of individual crops, a high degree of spatial aggregation in disease incidence was revealed.

Season-Long Dynamics of Spinach Downy Mildew Determined by Spore Trapping and Disease Incidence.

Peronospora effusa is an obligate oomycete that causes downy mildew of spinach. Downy mildew threatens sustainable production of fresh market organic spinach in California, and routine fungicide sprays are often necessary for conventional production. In this study, airborne P. effusa spores were collected using rotating arm impaction spore trap samplers at four sites in the Salinas Valley between late January and early June in 2013 and 2014. Levels of P. effusa DNA were determined by a species-specific quantitative polymerase chain reaction assay. Peronospora effusa was detected prior to and during the growing season in both years. Nonlinear time series analyses on the data suggested that the within-season dynamics of P. effusa airborne inoculum are characterized by a mixture of chaotic, deterministic, and stochastic features, with successive data points somewhat predictable from the previous values in the series. Analyses of concentrations of airborne P. effusa suggest both an exponential increase in concentration over the course of the season and oscillations around the increasing average value that had season-specific periodicity around 30, 45, and 75 days, values that are close to whole multiples of the combined pathogen latent and infectious periods. Each unit increase in temperature was correlated with 1.7 to 6% increased odds of an increase in DNA copy numbers, while each unit decrease in wind speed was correlated with 4 to 12.7% increased odds of an increase in DNA copy numbers. Disease incidence was correlated with airborne P. effusa levels and weather variables, and a receiver operating characteristic curve analysis suggested that P. effusa DNA copy numbers determined from the spore traps nine days prior to disease rating could predict disease incidence.

Three operational taxonomic units of Eimeria are common in Nigerian chickens and may undermine effective molecular diagnosis of coccidiosis.

BackgroundChicken is fast becoming the world’s most consumed meat. As a consequence poultry health is more important now than ever before, with pathogens of chickens recognised as serious threats to food security. One such threat are Eimeria species parasites, protozoa which can cause the disease coccidiosis. Eimeria can compromise economic poultry production and chicken welfare, and have serious consequences for poor livestock keepers. Seven Eimeria species that infect chickens are recognised with a global enzootic distribution. More recently three cryptic Operational Taxonomic Units (OTUx, y and z) have been described in populations of Eimeria recovered from chickens in Australia. Two of the three OTUs have also been detected in sub-Saharan Africa, but their occurrence, pathology and the risk they pose is largely unknown.ResultsNigeria has witnessed a dramatic expansion in poultry production and is now the largest poultry producer in Africa. Here, faecal samples collected from nine of 12 commercial chicken farms sampled in Kaduna state, Nigeria, were found to contain eimerian oocysts. After amplification by in vivo propagation all three cryptic OTU genotypes were detected using polymerase chain reaction (PCR), including OTUy for the first time outside of Australia. Comparison with a widely used, established Eimeria species-specific PCR assay revealed failure to detect the OTU genotypes.ConclusionsAll three of the Eimeria OTU genotypes appear to be common in north-western Nigeria. The failure of a leading species-specific molecular assay to detect these genotypes indicates a risk of false negative Eimeria diagnosis when using molecular tools and suggests that the spatial occurrence of each OTU may be far wider than has been recognised. The risk posed by these novel genotypes is unknown, but it is clear that a better understanding of Eimeria occurrence is required together with the validation of effective diagnostics.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-016-0713-9) contains supplementary material, which is available to authorized users.

Molecular Identification of an Invasive Wood-Boring Insect Lyctus brunneus (Coleoptera: Bostrichidae: Lyctinae) Using Frass by Loop-Mediated Isothermal Amplification and Nested PCR Assays.

Lyctus brunneus (Stephens) is one of the most destructive and worldwide invasive pests of seasoned woods for wooden products. This and other pest Lyctus species have had their distribution expanded by international and domestic human transportation of infested wood and wood products. Rapid detection and accurate identification of Lyctus species are effective tools for helping to eradicate them in new introduction sites. The accurate species-level identification of adults requires expert knowledge about their morphology. However, it takes much time and effort to recover suitable adult specimens because they are borers inside wood. Frass of Lyctus species can easily be detected and recovered in and around infested wood. Thus, frass was tested to see if it was a suitable sample to allow development of a rapid and technically easy molecular detection and identification method for L.brunneus. Species-specific primers were designed from the cytochrome c oxidase subunit I region of L.brunneus and used in development and testing of methods for successfully identifying them from their frass using the loop-mediated isothermal amplification (LAMP) or species-specific nested polymerase chain reaction (PCR) assays. The LAMP assay was faster and more sensitive for detecting the presence of DNA derived from L.brunneus in their frass than the nested PCR assay. These methodologies will be applicable for the rapid detection and identification of other wood-boring invasive pests in regulatory applications.

Occurrence of pathogenic Vibrio parahaemolyticus in crustacean shellfishes in coastal parts of Eastern India.

Aim:The objective of the study was to isolate and characterize pathogenic Vibrio parahaemolyticus from crustacean shellfishes (crab and shrimp) commonly retailed in coastal parts of eastern India.Materials and Methods:Samples were processed by bacteriological isolation followed by biochemical characterization in Kaper’s medium. Presumptively identified isolates were confirmed by species-specific Vp-toxR polymerase chain reaction (PCR) assay. Virulence and pandemic property of the confirmed V. parahaemolyticus isolates were determined by specific PCR assays.Results:On screening of 167 samples comprising crabs (n=82) and shrimps (n=85) by the standard bacteriological cultural method, V. parahaemolyticus was presumptively identified in 86.6% (71/82) and 82.3% (70/85) of respective samples. Of these, 46 (56%) and 66 (77.6%) isolates from crab and shrimp, respectively, were confirmed as V. parahaemolyticus by biochemical characterization (Kaper’s reaction) followed by specific Vp-toxR PCR assay. About 10 isolates each from crab and shrimp was found to carry the virulence gene (tdh). It denotes that 12.2% of crab and 11.7% of shrimp in the study area are harboring the pathogenic V. parahaemolyticus. Such tdh+ isolates (n=20) were subjected for screening of pandemic genotype by pandemic group specific (PGS) - PCR (PGS-PCR) and GS-PCR (toxRS gene) where 11 (6.5%) isolates revealed the pandemic determining amplicon (235 bp) in PGS-PCR and belonged to crab (7.3%) and shrimp (6%) samples; however, 2 (2.4%) isolates were positive in GS-PCR and belonged to crab samples only. These two GS-PCR+ isolates from crab were also positive in PGS-PCR.Conclusion:The findings of the study conclusively indicated that a considerable percentage of crab and shrimp in these areas were harboring pathogenic and pandemic V. parahaemolyticus posing a public health risk in consumption of improperly processed such shellfishes. Cross contamination of other marine and fresh water market fishes by such shellfishes in these areas may provide scope for spreading this pathogen in community food chain.

English

Lentinula edodes is among the five globally cultivated edible mushrooms, which are wood decaying spore bearing Basidiomycetes possessing separate hyphae. Specific identification of this fungus from others in the division Basidiomycota using specific primers enables a fast and accurate detection through polymerase chain reaction (PCR). As a prelude to additional nutritional and sequence characterization research, we have developed a species-specific PCR assay for this fungus after screening four primer-pairs and two universal primer pairs. The primer-pair LE1F/R was specific in amplifications of ATCC-defined L. edodes strains and did not amplify DNA from six medicinally and nutritionally important fungal reference strains, Oyster (Pleurotus ostreatus), Maitake (Grifola frondosa), Enoki (Flammulina velutipes), Baby bella (Agaricus bisporus), Porcini (Boletus edulis), and Chanterelle (Cantharellus cibarius). However, amplifications using the universal primers were positive for all six strains. This assay will therefore serve to validate morphology-based-identifications of L. edodes strains. Key words: Lentinula edodes, LE1F/R, species-specific primers.

First detection and molecular identification of Babesia vogeli from Rhipicephalus sanguineus (Acari: Ixodidae) in Taiwan

A total of 578 Rhipicephalus sanguineus ticks collected from dogs in Taiwan were examined for Babesia by species-specific polymerase chain reaction assay based on the 18S small subunit ribosomal RNA (ssrRNA) gene. Babesia DNA was detected in 1.04 % (6/578) of Rh. sanguineus ticks. Phylogenetic analysis revealed that these Babesia spp. were genetically linked to the same clade within the genospecies of Babesia vogeli and could be discriminated from other genospecies of Babesia. Intra-species analysis based on the genetic distance values indicated a lower level (0.079) compared with other genospecies of Babesia (GD > 0.094) and out-group protozoa (GD > 0.236). This study provides the first molecular evidence of B. vogeli detected and identified in various stages of Rh. sanguineus ticks removed from dogs in Taiwan. Detection of Rh. sanguineus in flat male ticks may imply the possible mechanism of transstadial transmission in Rh. sanguineus ticks. The vector competence and the diversity of Babesia species harbored by Rh. sanguineus ticks need to be further investigated.