Species-specific Forward Primers Research Articles

Development of rapid and cost-effective multiplex PCR assays to differentiate catfish of the genus Brachyplatystoma (Pimelodidae-Siluriformes) sold in Brazil.

The catfishes Brachyplatystoma filamentosum (Kumakuma), Brachyplatystoma vaillantii (Laulao catfish), and Brachyplatystoma rousseauxii (gilded catfish) are important fishery resources in Brazil, where they are sold both fresh and in the form of fillets or steaks. These species have morphological similarities, thus, they can be easily misidentified or substituted, especially after processed. Therefore, accurate, sensitive, and reliable methods are needed for the identification of these species to avoid commercial fraud. In the present study, we develop two multiplex PCR assays for the identification of the three catfish species. Each multiplex protocol combined three species-specific forward primers and a universal reverse primer to produce banding patterns able to discriminate the target species unequivocally. The length of the cytochrome C oxidase subunit I (COI) fragments was approximately 254 bp for B. rousseauxii, 405 bp for B. vaillantii, and 466 bp for B. filamentosum, while the control region (CR) assay produced fragments of approximately 290 bp for B. filamentosum, 451 bp for B. vaillantii, and 580 bp for B. rousseauxii. The protocols were sensitive enough to detect the target species at a DNA concentration of 1 ng/µL, with the exception of the CR of B. vaillantii, in which the fragment was only detectable at 10 ng/µL. Therefore, the multiplex assays developed in the present study were sensitive, accurate, efficient, rapid, and cost-effective for the unequivocal identification of the target species of Brachyplatystoma. They can be utilized by fish processing industries to certify their products, or by government agencies to authenticate products and prevent fraudulent commercial substitutions.

First report of Fusarium fujikuroi causing root rot and seedling elongation of soybean in Indiana.

In summer 2020, 127 soybean (Glycine max (L.) Merr) seedlings (V1-V3 stage) showing reduced vigor or crown lesions were collected at Purdue's Agronomy Center for Research and Education in West Lafayette, Indiana. Root tissues from two seedlings with necrotic cotyledons and root rot were surface-sterilized and plated on dichloran-chloramphenicol-peptone agar (Andrews and Pitt 1986). Emerging hyphal tips were transferred to potato dextrose agar (PDA). Single-spore cultures were obtained and grown on PDA. Both isolates developed floccose white aerial mycelia with reddish-pink coloration in the media in 2 weeks on the benchtop. On carnation leaf agar, macroconidia formed on orange sporodochia within 2 weeks in darkness at 25C. Macroconidia were 3-5 septate, measuring 26 - 41 × 2.5 - 3.7 μm (avg. 34.8 × 3.2 μm, n=40). Microconidia were abundant in chains and false heads forming on both mono- and polyphialides, and measured 2.5 - 8.75 x 2.5 μm (avg. 5.9 × 2.5 μm, n=40). These characteristics were consistent with species descriptions of F. fujikuroi [Sawada] Wollenw. (teleomorph Gibberella fujikuroi) (Leslie and Summerell 2006). DNA was extracted from mycelium and the following genes were amplified and sequenced: the internal transcriber spacer (ITS) region using ITS1/ITS4 primers (White et al. 1990) (GenBank accessions MW463362/MW463363), the mitochondrial small subunit (mtSSU) rDNA using MS1/MS2 primers (White et al. 1990) (MW465310/MW465307), and the partial translation elongation factor 1-alpha (TEF1α) gene using 983F/1567R primers (Rehner and Buckley 205) (MW475297/MW475298). In GenBank BLAST searches, these sequences showed 100% identity to both F. proliferatum and F. fujikuroi. Species-specific forward primers Fuji1F and Proli1F were then used in combination with reverse primer TEF1R to amplify another region in the TEF1α gene (Amatulli et al. 2012). Proli1F/TEF1R primers failed under a variety of annealing temperatures while Fuji1F/TEF1R primers succeeded, and the products were sequenced (MW475299/MW475300). GenBank BLAST searches revealed 100% identity of both isolates to F. fujikuroi (MT448248.1). A pathogenicity test was conducted with isolate AC13 in the greenhouse following the protocol of (Ellis et al. 2013). Ten seeds (cv. Williams) each were used for inoculation and control, respectively, with one seed per cup. Root rot symptoms similar to those observed in the field were observed 14 days after planting on all inoculated plants but not on controls (VC stage). Infected plants showed symptoms of pre-emergence damping off, reddish-brown lesions on the tap and lateral roots, and root necrosis. Three plants also exhibited hyper-elongation of the stem (12.5, 11.1, and 18 cm, vs controls: avg. 6.8 cm, max. 8.5 cm, stdev 0.78 cm). F. fujikuroi was successfully reisolated from inoculated plants but not from controls and identified as described above. F. fujikuroi has been reported causing soybean root rot in China (Zhao et al. 2020), Korea (Choi et al. 2019), and the state of Kansas (Pedrozo et al. 2015). To our knowledge this is the first report of F. fujikuroi infecting soybeans in the state of Indiana. F. fujikuroi is known to cause elongated seedlings in rice (Leslie and Summerell 2006). Pedrozo et al. (2015) reported that F. fujikuroi isolated from soybean caused seedling elongation in rice but not in soybean. The increased distribution and new host symptomology observed here warrants heightened attention for the control of this pathogen.

First report of 'Candidatus Liberibacter solanacearum' on carrot in Serbia.

At the beginning of July 2020, three-month-old carrot plants (Daucus carota L. variety Maestro F1) grown in a commercial field 1.2 ha in size at the Begeč locality (45°14'30.38" N 19°36'44.82" E) in southern part of the Bačka region, Vojvodina, Serbia, exhibited symptoms of yellowing and reddish leaf discoloration. At the end of July, leaves on the infected plants became bronze and purplish, while their shoots and roots were stunted due to dehydration, with pronounced proliferation. In some cases, the damage was so extensive that it led to plant decay. The disease incidence of 0.5-1% recorded early in July rapidly escalated, reaching 10-15% in the first ten days of August. The observed symptoms resembled those caused by 'Candidatus Liberibacter solanacearum' (CaLso), a phloem-limited proteobacterium (1). To detect and identify CaLso, 15 symptomatic diseased and 5 asymptomatic healthy carrot plants were subjected to conventional polymerase chain reactions (PCR) using two primer sets specific to CaLso, and positive PCR products were further sequenced using commercial facilities (Macrogen Europe). Total DNA was extracted from petiole and root tissues using a commercial kit (Qiagen DNEasy Plant Mini Kit) following the manufacturer-recommended protocol. In the first PCR, using the Lso TX 16/23 F/R primer pair that targets the 16S-23S rRNA IGS region specific to CaLso (2), all 15 diseased samples yielded a band of 383 bp size. After sequencing, 100% homology was noted among tested isolates; therefore, one isolate coded as 1842/20 was chosen as representative and was deposited in NCBI GenBank under Accession number MT948144. BLAST analysis showed 99.70% identity of Serbian carrot isolates with those of the CaLso isolate 80022 originating from celery seed in Slovenia or Italy (Acc. no. KY619977) (3), as well as 99.41% identity with isolate GBBC_Clso_03 from carrot in Belgium (Acc. no. MH734515) and 98.22% identity with the sequence of the CaLso reference strain NZ082226 (Acc. no. EU834130) isolated from tomato in New Zealand (4). In the second PCR, species-specific forward primer LsoF empirically designed at the signature region of the 16S rRNA sequence of CaLso (5) in combination with the universal liberibacter reverse primer OI2c (6) yielded a target of 1163 bp size in all 15 diseased symptomatic carrot samples. Representative isolate 1842/20 was deposited in NCBI GenBank under Acc. no. MW187524. Based on the nucleotide BLAST analysis, the sequence of Serbian carrot isolate showed 100% identity with CaLso strains 16-004 and 16-011 originating from carrot in Finland (Acc. no. MG701014 and MG701015, respectively) and 99.64% identity with CaLso reference strain NZ082226 (Acc. no. EU834130). Five healthy asymptomatic carrot plant samples were negative for the presence of CaLso in both PCR tests employed in this work. To our knowledge, this is the first report of CaLso causing the disease in carrot in Serbia. These results suggest a wider distribution of this pathogen than previously reported in Europe. In 2014, Psyllid Bactericera trigonica (Hemiptera, Triozidae) was described for the first time as a potential vector for CaLso transmission in few localities, including Begeč (7). Considering that its vectors are presently unidentified, certain aspects of CaLso genomics, diversity, epidemiology and vector dynamics will be studied further in future investigations.

CML-004: Imatinib is Protective Against Falciparum Malaria: A Case Control Study from a Tertiary Care Centre in West Bengal

Context Rising evidence of artemisin resistant malaria in Indian subcontinent and South America has prompted researchers to search for alternative antimalarials. In the last few decades, few reports and subsequent in vitro results have emerged showing dependence of malarial parasites, particularly P falciparum, on RBC tyrosine kinases for its maturation and egress. As there were no clinical data to support the hypothesis, we conducted a case control study with patients of CML-CP on fixed dose imatinib (TKI) as cases and a representative healthy population as control. Objective To compare the prevalence of asymptomatic falciparum and vivax infections in study cases with the prevalence of matched control taken from healthy blood donors. Methods A standard questionnaire and rapid diagnostic test (RDT) for plasmodium antigens from the blood of cases attending CML clinic were used as study tools. 191 CML patients who are on imatinib 400mg/d for a minimum period of 1 year were analysed. Controls were healthy voluntary blood donors attending the hospital during the same time-frame. Study time was April-September (peak malaria season). All RDT +ve cases were internally validated with PCR using two species specific forward primers. Results Out of 54 febrile episodes among cases during the study period, one had vivax malaria. RDT testing on non-febrile participants revealed two samples positive for dual vivax-falciparum antigens and the other two for vivax antigen. PCR showed three cases positive for vivax and one positive for both vivax and falciparum. Among 205 controls, three had vivax infections, three had falciparum infections and four were found to have infections with both. The concordance for positive results among the diagnostic modalities (RDT and PCR) was found to be 93.3%. The prevalence of malarial infections in cases and controls were found to be 2.05% and 4.87% respectively while falciparum parasitemia in cases and controls were found to be 0.51% and 3.41% respectively. Conclusion Analysis showed that the difference of asymptomatic falciparum carriage among cases and control was statistically significant suggesting a protective effect of imatinib against falciparum infections. The association of vivax positivity with imatinib exposure was however not statistically significant.

Meat Species Identification: Amplification Refractory Mutation System-Polymerase Chain Reaction–Based Assay

True verification of meat species is critical for religious, economical, legal, and/or public health concerns. Current methods available for verification of meat species are very time-consuming and/or costly. So considering the necessity, a novel polymerase chain reaction assay (PCR)–based ARMS (amplification refractory mutation system) type identification assay for detection of 6 most common mammalian species—Equus asinus (donkey), Equus caballus (horse), Bos Taurus (cow), Bubalus bubalis (buffalo), Capra hircus (goat), and Canis lupus familiaris (dog)—was described in present study by designing species-specific forward primers against variable regions and a single universal reverse primer against a very conserved region of nuclear beta actin (ACTB) gene by investigation of areas of homology and variation. As compared with conventional designing of primers, ARMS type primer designing is a better alternative as it allows more room for choice and offers differentiation of closely related species by exploiting just a single nucleotide base difference. PCR bands of 128, 229, 273, 362, 710, and 796 bp were generated on electrophoretic gel for buffalo, donkey, cow, horse, dog, and goat meat species respectively. Besides singleplexing, duplex (multiplex) PCR for donkey and horse, donkey and goat, and donkey and buffalo were also performed which successfully generated corresponding bands. The method is a simple and straightforward setup; results can be interpreted easily in a short time and do not need validation by sequencing. Results of the present study clearly demonstrate that the method can be used as an identification tool for differentiation between cow, buffalo, goat, donkey, horse, and dog species.

Comparison of Two Multiplex PCR Systems for Meat Species Authentication

Background: Meat species adulteration has become a problem of concern. This study aimed to compare two previously published multiplex Polymerase Chain Reaction (PCR) methods for meat species authentication. Methods: The primers used in the first multiplex PCR involved species-specific reverse primer for sheep, goat, cattle, pig, and donkey with universal forward primer. In the second multiplex PCR, the primers included species-specific forward and reverse primer for pork, lamb, ostrich, horse, and cow. The extracted DNA was then amplified with species-specific primers and with mix primers separately in the respective multiplex PCR. Results: The first multiplex PCR was accompanied with cross reactivity, whereas the second multiplex PCR was specific as expected for pork, lamb, ostrich, horse, and cow. The first set of multiplex PCR showed not always amplification of all species-specific DNAs with a mixture of DNA from mentioned animals. Regarding the second set of primers, the extracted DNA of different meat species was amplified with corresponding species primers as simplex PCR resulting in specific amplicons for species DNA prepared from sheep, ostrich, horse, pig, and cattle with the specific PCR products of 119, 155, 253, 100, and 311 bp, respectively. Conclusion: Based on the present investigation, we recommend the multiplex PCR with the second set of primers included species-specific forward and reverse primers for species authentication of five meat types, including pork, lamb, ostrich, horse, as well as cow.

A sensitive and a rapid multiplex polymerase chain reaction for the identification of Candida species in concentrated oral rinse specimens in patients with diabetes

Objectives: Oral candidiasis is being frequently recognized in patients with diabetes, and is associated with multiple pathogens including Candida albicans, Candida parapsilosis, Candida glabrata and Candida tropicalis. The aim of this study was to evaluate a usefulness of a Multiplex Polymerase Chain Reaction as a rapid diagnostic tool for identification of four oral Candida pathogens in patients with diabetes.Materials and methods: A multiplex PCR was optimized to identify four Candida species in concentrated oral rinse samples. Common reverse primer, ITS4 and four species-specific forward primers targeting ITS1 and ITS2 regions of yeast genome were used. Species-specific single amplicon were detected by agarose gel electrophoresis. Performance efficacy of multiplex PCR was compared with phenotypic identification.Results: Out of 100 oral rinse samples, 72 were culture positive and of these 43 were at risk of oral Candida infection (>600cfu/ml). Multiple Candida species including C. albicans, C. parapsilosis and C. tropicalis were identified in 22 samples which had risk of oral Candida infection. In total, 85 patients were positive for Candida by multiplex PCR and of them 49 had multiple Candida species. All 43 colonized specimens were also positive by multiplex PCR. C. albicans was the most predominant organism (75/85) followed by C. parapsilosis (47/85), C. tropicalis (17/85) and C. glabrata (6/85). In specimens with multiple species, the two most common organisms were C. albicans and C. parapsilosis. Multiplex PCR yielded a sensitivity of 10 Candida cells/ml of oral rinse sample.Conclusions: Multiplex PCR is found to be rapid, sensitive and specific than phenotypic identification methods in discriminating multiple Candida species in oral rinse specimens.

Diversity of root-lesion nematodes (Pratylenchus spp.) associated with wheat (Triticum aestivum and T. durum) in Morocco

Root-lesion nematodes (Pratylenchus) have a worldwide distribution and cause severe production constraints on numerous important crops. During a survey of the wheat-growing area of Morocco, 17 populations of root-lesion nematode were collected. They were identified on the basis of their morphological and morphometric characters, and by molecular methods. Microscopic observations of females and males demonstrated the occurrence ofP. penetransin 13 of the 17 samples;P. thorneiandP. pseudocoffeaewere detected in four samples from Zaers and a single sample from Settat, respectively. A duplex PCR primer set was used to confirm the presence ofP. penetranswhile the species-specific forward primer PTHO and the common reverse primer D3B were used forP. thornei. For the remaining populations, the D2-D3 expansion segments of the 28S rRNA gene were amplified and the obtained sequences were compared with those ofPratylenchusspecies in the GenBank database. This comparison confirmed the morphological identifications and revealed a population ofP. pinguicaudatus. The study of the phylogenetic relationship of the MoroccanPratylenchuspopulations showed a high similarity (99-100%) between allP. penetranspopulations. The population dynamics of sixPratylenchuspopulations from Morocco were evaluated on carrot disk cultures at 4, 8 and 12 weeks after inoculation, and at 10, 15, 20 and 25°C. The optimum temperature for reproduction of all populations was 20°C. After 8 weeks at this temperature, nematode numbers increased up to 458-fold, 310-fold and 252-fold for the four populations ofP. penetrans, theP. thorneiand theP. pseudocoffeaepopulation, respectively.

Simultaneous detection of the feline lungworms Troglostrongylus brevior and Aelurostrongylus abstrusus by a newly developed duplex-PCR

In addition to Aelurostrongylus abstrusus (Strongylida: Angiostrongylidae), referred to as the feline lungworm, Troglostrongylus brevior (Strongylida: Crenosomatidae) has recently been identified as an agent of bronco-pulmonary infestations in cats. These two parasites have a similar biology, share ecological niches, potentially co-infesting cats, but are difficult to be differentiated due to the morphological similarities of their first-stage larvae (L1). This paper describes a molecular tool, based on single-step duplex polymerase chain reaction (duplex-PCR) on the ribosomal internal transcribed spacer 2 region (ITS-2) for the simultaneous detection and differentiation of T. brevior and A. abstrusus. L1 of both species were collected from faecal samples, morphologically identified, and single larval specimens isolated. An aliquot of faeces was used as a test sample for a case of mixed natural infestation. The duplex-PCR was performed using species-specific forward primer sets for the ITS-2 region (i.e., A. abstrusus: 220bp; T. brevior: 370bp). The detection limit of the molecular assay was also assessed by serial dilutions of DNA from single larvae of both species (from ∼4.0 to 4.0×10−5μg/μl). The duplex-PCR carried out on individual DNA samples was able to detect as low as 5.2×10−3μg/μl of DNA for A. abstrusus, 4.9×10−3μg/μl for T. brevior, and as low as 4.0×10−3μg/μl for samples containing both species. Species-specific bands of the expected sizes and two bands were simultaneously amplified from the faecal sample containing both species. The phylogenetic analyses of the ITS-2 sequences here examined and those available for other metastrongyloids were concordant in clustering them with those of other Troglostrongylus brevior and A. abstrusus sequences available in GenBank database. This molecular approach proved to be effective and highly sensitive for the simultaneous detection of the two lungworms species and it might be used for molecular epidemiological studies and for monitoring therapeutic protocols.

Development and Evaluation of a Single-Step Duplex PCR for Simultaneous Detection of Fasciola hepatica and Fasciola gigantica (Family Fasciolidae, Class Trematoda, Phylum Platyhelminthes)

A single-step multiplex PCR (here referred to as a duplex PCR) has been developed for simultaneous detection and diagnosis of Fasciola hepatica and F. gigantica. These species overlap in distribution in many countries of North and East Africa and Central and Southeast Asia and are similar in egg morphology, making identification from fecal samples difficult. Based on a comparative alignment of mitochondrial DNA (mtDNA) spanning the region of cox1-trnT-rrnL, two species-specific forward primers were designed, FHF (for F. hepatica) and FGF (for F. gigantica), and a single reverse primer, FHGR (common for both species). Conventional PCR followed by sequencing was applied using species-specific primer pairs to verify the specificity of primers and the identity of Fasciola DNA templates. Duplex PCR (using three primers) was used for testing with the DNA extracted from adult worms, miracidia, and eggs, producing amplicons of 1,031 bp for F. hepatica and 615 bp for F. gigantica. The duplex PCR failed to amplify from DNA of other common liver and intestinal trematodes, including two opisthorchiids, three heterophyids, an echinostomid, another fasciolid, and a taeniid cestode. The sensitivity assay showed that the duplex PCR limit of detection for each Fasciola species was between 0.012 ng and 0.006 ng DNA. Evaluation using DNA templates from 32 Fasciola samples (28 adults and 4 eggs) and from 25 field-collected stools of ruminants and humans revealed specific bands of the correct size and the presence of Fasciola species. This novel mtDNA duplex PCR is a sensitive and fast tool for accurate identification of Fasciola species in areas of distributional and zonal overlap.

Species specific primers for identification of Alternaria solani, in combination with analysis of the F129L substitution associates with loss of sensitivity toward strobilurins

A species specific forward primer was designed in order to identify the phytopathogenic fungus Alternaria solani, which causes early blight on potato. The reverse primer came from another survey of the American population of A. solani. The combination of these two primers can be used both as molecular identification of A. solani as well as sending the PCR product for sequencing in search for the F129L substitution associated with loss of sensitivity toward fungicides based on strobilurins. The diagnostic method has been successfully used when identifying the causal agent of lesions on potato leaflets during large field assays.

A multiplex PCR for the simultaneous detection of species of filarioids infesting dogs

The present study reports the applicability of a multiplex PCR for the simultaneous detection and differentiation of common filarioids infecting dogs, i.e., Dirofilaria immitis, Dirofilaria repens, Acanthocheilonema reconditum and Cercopithifilaria sp. Amplicons of different sizes (i.e., 170bp, 480bp, 590bp and 300bp, respectively) of regions within the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were amplified on a single-step multiplex PCR using a mix of species-specific forward primers coupled with a single reverse primer. Experiments were carried out by amplifying genomic DNA extracted from blood or skin samples test-positive for microfilariae (mff). The number of mff present in each blood sample was quantified (from 800 to 25,000mff/ml for A. reconditum and D. repens, respectively) and mixed blood samples were tested for the simultaneous detection of DNA from these mff. Specific amplicons for blood-circulating mff of A. reconditum, D. immitis and D. repens and for those whose adults are localized in skin (i.e., A. reconditum and Cercopithifilaria sp.) were simultaneously detected on agarose gel up to a dilution of 250mff/ml for D. repens. The specific identity of the amplicons was confirmed by sequencing. The multiplex PCR assay reported herein represents a new tool for the molecular detection and differentiation of canine filarioids in blood and skin samples.

Species distribution and in vitro fluconazole susceptibility of clinical Candida isolates in a Brazilian tertiary-care hospital over a 3-year period

In this study, we aimed at identifying Candida isolates obtained from blood, urine, tracheal secretion, and nail/skin lesions from cases attended at the Hospital Universitário de Londrina over a 3-year period and at evaluating fluconazole susceptibilities of the isolates. Candida isolates were identified by polymerase chain reaction (PCR) using species-specific forward primers. The in vitro fluconazole susceptibility test was performed according to EUCAST-AFST reference procedure. Isolates were obtained from urine (53.4%), blood cultures (19.2%), tracheal secretion (17.8%), and nail/skin lesions (9.6%). When urine samples were considered, prevalence was similar in women (45.5%) and in men (54.5%) and was high in the age group >61 years than that in younger ones. For blood samples, prevalence was high in neonates (35%) and advanced ages (22.5%). For nail and skin samples, prevalence was higher in women (71.4%) than in men (28.6%). Candida albicans was the most frequently isolated in the hospital, but Candida species other than C. albicans accounted for 64% of isolates, including predominantly Candida tropicalis (33.2%) and Candida parapsilosis (19.2%). The trend for non-albicans Candida as the predominant species was noted from all clinical specimens, except from urine samples. All Candida isolates were considered susceptible in vitro to fluconazole with the exception of isolates belonging to the intrinsically less-susceptible species C. glabrata. Non-albicans Candida species were more frequently isolated in the hospital. Fluconazole resistance was a rare finding in our study.

Validation of a four-primer real-time PCR as a diagnostic tool for single and mixed Plasmodium infections

Although microscopy remains the reference standard for malaria diagnosis, molecular tools are attracting increasing interest. To improve the detection of mixed infections, we developed a four-primer real-time PCR with four Plasmodium species-specific forward primers, based on the pan-primer design with universal Plasmodium primers as described previously. After validation for analytical sensitivity, specificity and reproducibility, the four-primer PCR was evaluated on 351 blood samples from patients presenting at the outpatient clinic of the Institute of Tropical Medicine (Belgium). With the four-primer PCR, we identified 188 Plasmodium falciparum (Pf), 54 Plasmodium vivax (Pv), 52 Plasmodium ovale (Po) and 13 Plasmodium malariae (Pm) single infections, 27 mixed infections (14 Pf + Pm; 12 Pf + Po; one Pv + Pm) and 17 negative specimens. We found lower cycle threshold values than with the pan-primer PCR, with a mean difference of 2.23, a higher analytical sensitivity (in asexual parasites/µL: Pf/Pv, 0.02; Po, 0.004; Pm, 0.006) and 15 extra mixed infections. As compared with microscopy, 17 extra mixed infections were detected and Plasmodium species were identified in four microscopy-positive samples in which species identification was not possible. Additionally, the PCR corrected 13 species mismatches between Po and Pv, and in 11 cases detected Pf as a second species that was not identified by microscopy and in five of them was not detected by rapid diagnostic tests (RDTs). PCR confirmed the presence of Pf in 30/46 histidine-rich protein-2-positive samples that were microscopy-negative. We conclude that the presently developed four-primer real-time PCR is complementary to standard malaria diagnostic tests in clinical laboratories, with an added value for simultaneous identification of the four Plasmodium species and the detection of mixed infections.

A diagnostic multiplex polymerase chain reaction method to identify Japanese internal apple-feeding Lepidopteran pests: Grapholita molesta, Grapholita dimorpha (Lepidoptera: Tortricidae), and Carposina sasakii (Lepidoptera: Carposinidae)

We present a multiplex polymerase chain reaction method to differentiate between three Japanese internal apple-feeding pests: the oriental fruit moth, Grapholita molesta (Busck), Grapholita dimorpha Komai, and the peach fruit moth, Carposina sasakii Matsumura. A 1,342-bp fragment of mitochondrial cytochrome oxidase subunit I (COI) was sequenced in each species. The three species showed consistent and diagnostic differences in the region of the COI gene, from which three species-specific forward primers were designed. The forward primers along with one universal reverse primer were used to selectively amplify DNA from specimens of diverse geographic origin for each corresponding target species. This method enabled easy, immediate, and accurate identification of internal feeding Lepidoptera in apples and other fruits.

Development of SCAR primers for the detection of Cadophora luteo-olivacea on kiwifruit and pome fruit and of Cadophora malorum on pome fruit.

800x600 Normal 0 14 false false false IT X-NONE X-NONE MicrosoftInternetExplorer4 In recent years a postharvest disease of kiwifruit, characterized by skin pitting appearing after 3 or more months of storage, and caused by C. luteo-olivacea , has been reported in most Italian packinghouses. Forty-four strains isolated from kiwifruit harvested in Italy during the period 2001-2009 were tested for their pathogenicity on kiwifruit, apple and pear. The pathogenicity tests showed that different isolates of C. luteo-olivacea were pathogenic on the three fruit species stored at 1°C for 120 days with various degree of virulence. A PCR-based method to identify the pathogen species in kiwifruit was developed. Sequencing of the internal transcribed spacer (ITS1, 5.8S gene and ITS2) region of the rDNA (ITS) showed high similarity with the species C. malorum , agent of side rot of apples and pears. Variation within the ITS was used to design one reverse primer common to both species and two species specific forward primers to specifically identify isolates of C. luteo-olivacea and C. malorum. Both SCAR (sequence characterized amplified region) primer pairs showed to be specific for either Cadophora species when cross-tested and assessed on other species of Cadophora or on species of typical postharvest pathogens of kiwifruit, apple and pear .

Methods for detection and differentiation of existing and new crinivirus species through multiplex and degenerate primer RT-PCR

A method was developed for rapid identification and differentiation of both known and novel crinivirus species involving both multiplex and degenerate reverse transcription-polymerase chain reaction (RT-PCR). The multiplex method can discriminate among known criniviruses infecting vegetable and small fruit crops, and rapidly identify viruses associated with disease symptoms, as well as identification of mixed crinivirus infections. Four host groups for multiplex detection of criniviruses were selected based on the types of crops where specific criniviruses would be expected to occur. Each detection group contained three to four crop-specific primers designed to the same region of the gene encoding the highly conserved RNA-dependent RNA polymerase gene (RdRp) of criniviruses for rapid, single-reaction determination of which crinivirus(es) may be infecting a plant. Degenerate reverse primers used for RT and in PCR were designed to amplify all members of each host group, and were coupled with species-specific forward primers resulting in four separate single-reaction cocktails for detection of most criniviruses sequenced to date, whether present in single or mixed virus infections. Additional viruses can be added to multiplex detection by adjustment of primer concentration for balanced detection of target viruses. In order to identify unknown putative criniviruses or those for which sequence information is not yet available, a genus-wide, universal degenerate primer set was developed. These primers also targeted the crinivirus RdRp gene, and amplify a wide range of crinivirus sequences. Both detection systems can be used with most RNA extraction methods, and with RT-PCR reagents common in most laboratories.

First Report of Brown Rot of Stone Fruit Caused by Monilinia fructicola in Italy

Monilinia fructicola, causal agent of brown rot, is one of the most important fungal pathogens of stone fruit. M. fructicola is a quarantined pathogen in Europe. During the summer of 2008 in 15 orchards located in Piedmont (northern Italy), 12,500 stone fruits (cherries, apricots, peaches, nectarines, and plums) were stored in cold chambers at 4 and 6°C and monitored for 8 weeks for the presence of Monilinia spp. M. fructicola was detected on 0.5% of nectarines (cvs. Sweet Red and Orion) that originated from two orchards in Lagnasco. Symptoms appeared on the fruit during storage, starting 3 weeks after harvest. Fruit rot lesions were brown, sunken, and covered with grayish tufts. The majority of infected fruit became dry and mummified. Brown rot symptoms were similar to those caused by endemic M. fructigena and M. laxa. Symptoms began with a small, circular, brown spot, and the rot spread rapidly. At the same time, numerous, small, grayish stromata developed. Finally, the whole surface of the fruit was covered by conidial tufts. Tissues were excised from diseased stone fruits and cultured on potato dextrose agar (PDA) amended with 25 μg of streptomycin per liter. The isolates produced abundant mycelium on PDA at 20 ± 2°C. Colonies were initially gray, but after sporulation, they became hazel, showing concentric rings (sporulation is sparse in M. laxa or M. fructigena). Conidia were one-celled, ellipsoid, hyaline, 15.2 × 10.1 μm, and produced in branched monilioid chains (2). Preliminary morphological identification of fungi resembling M. fructicola was confirmed by PCR using genomic DNA extracted from the mycelia of pure cultures. The DNA was amplified with a common reverse primer and three species-specific forward primers (3) obtained from a sequence characterized amplified region and a product of 535 bp, diagnostic for the species M. fructicola, was obtained. BLAST analysis of the amplified sequence (GenBank Accession No. FI569728) showed 96% similarity to the sequence of a M. fructicola isolated from Canada (GenBank Accession No. AF506700), 15% similarity to M. laxa ATCC11790 (GenBank Accession No. AF506702), and 35% similarity to a M. fructigena sequence isolated in Italy (GenBank Accession No. AF506701). Moreover, two sequences obtained through the amplification of ribosomal region ITS1-5.8S-ITS2, showing 100% similarity to the same ribosomal sequence of M. fructicola, were deposited in GenBank (Accession Nos. FJ411109 and FJ411110). The pathogen was detected on some mummified fruit from the same orchards in November of 2008. Pathogenicity was tested by spraying 103 conidia/ml on 10 surface-sterilized artificially wounded nectarines per strain of M. fructicola. After 5 days of incubation at 20 ± 2°C, typical, brown, rot symptoms developed on inoculated fruit. M. fructicola was reisolated from the inoculated fruit on PDA. Symptoms did not appear on control fruit. To our knowledge, this is the first report of M. fructicola in Italy. Its occurrence in Europe has been reported sporadically in Austria and France, and in 2006, it was detected in Hungary and Switzerland on peaches and nectarines imported from Italy and Spain (1,4).

Multiplexed Real-Time PCR Assay for Discrimination of Plasmodium Species with Improved Sensitivity for Mixed Infections

The implementation of real-time PCR for the diagnosis of malaria has been hampered by poor sensitivity for the detection of mixed infections. We have optimized a method that enhances the sensitivity of detection of minor species in mixed infections within a single multiplex reaction. Our assay uses species-specific forward primers in combination with a conserved reverse primer and largely overcomes primer competition for the minor species DNA. With a blind panel of clinical samples, we successfully identified the species in 13/16 mixed infections. This assay was further validated with 91 blood samples and demonstrated a specificity and sensitivity for single infections of 100% compared with nested PCR as the "gold standard." This test has been implemented for routine confirmation of malaria species in Alberta, Canada. In comparison with species identification by microscopy, the real-time PCR test demonstrated greater sensitivity for the identification of species causing low-level and mixed infections and for the discrimination of Plasmodium species other than Plasmodium falciparum. Our experience supports a role for real-time PCR in the identification of malarial species in conjunction with microscopy.

Prevalence of Porphyromonas gingivalis and Bacteroides forsythus in Chronic Periodontitis by Multiplex PCR

The present research decided to study prevalence of Porphyromonas gingivalis and Bacteroides forsythus in chronic periodontitis patient by use of Multiplex PCR. The subgingival plaque samples from 61 patients suffering from chronic periodontitis with probing depth PD > or = 6 and 40 healthy controls were collected by sterile curette. In this study we used two species-specific Forward primers in combination with a single Reverse primer. These primers target variable and conserved region of 16S rRNA gene, respectively. The study included 61 patients (34 women, 27 men; 24-69 years of age; mean 43) and 40 periodontally healthy controls (22 Women, 18 men, 21-69 years in age; mean 41.35%). Porphyromonas gingivalis was detected in 51 samples (83.61%) and 16 samples (40%) of chronic periodontitis patients and healthy subjects, respectively and Bacteroides forsythus was detected in 32 samples (52.50%) of chronic periodontitis patients and was not detected in any sample from healthy persons. We set up Multiplex PCR in order to detect P. gingivalis and B. forsythus simultaneously. The present data suggest that P. gingivalis is a more important cofactor in etiology of chronic periodontitis. Further studies are needed to determine spectrum of pathogenicity of the disease and effective management of diagnosis and treatment in order to decrease the risk of periodontic complicates such as systemic infection.