The development of multiplex PCR assays for the rapid identification of multiple Saccostrea species, and their practical applications in restoration and aquaculture.

BACKGROUND: The ecology and biology of oysters (Ostreidae) across the tropics is poorly understood. Morphological plasticity and shared characteristics among oysters have resulted in the misidentification of species, creating challenges for understanding basic species-specific biological information that is required for restoration and aquaculture. Genetic barcoding has proven essential for accurate species identification and understanding species geographic ranges. To reduce the costs of molecular species identification we developed multiplex assays using the cytochrome c oxidase subunit I (COI or cox1) barcoding gene for the rapid identification of five species of oysters within the genus Saccostrea that are commonly found in Queensland, Australia: Saccostrea glomerata, Saccostrea lineage B, Saccostrea lineage F, Saccostrea lineage G, and Saccostrea spathulata (lineage J). RESULTS: Multiplex assays were successful in species-specific amplification of targeted species. The practical application of these primers was tested on wild spat collected from a pilot restoration project in Moreton Bay, Queensland, with identified species (S. glomerata, lineage B and lineage G) validated by Sanger sequencing. DNA sampling by extraction of oyster pallial fluid was also tested on adult oysters collected from the Noosa estuary in Queensland to assess whether oysters were able to be identified non-destructively. DNA concentrations as low as 1 ng/ µL still amplified in most cases, allowing for identification, and mortality at 6 weeks post pallial fluid collection was low (3 out of 104 sampled oysters). CONCLUSION: These multiplex assays will be essential tools for species identification in future studies, and we successfully demonstrate their practical application in both restoration and aquaculture contexts in Queensland. The multiplex assays developed in this study outline easily replicable methods for the development of additional species-specific primer sets for the rapid identification of other species of Saccostrea found across the Indo-Pacific, which will be instrumental in unravelling the taxonomic ambiguities within this genus in tropical regions.

Confirmatory Assay for Laboratory Diagnosis of Malaria Using Molecular Approach.

BACKGROUND: Prompt malarial treatment and surveillance is crucial for accurate diagnosis of Plasmodium Sp. Gold standard microscopic examination has been widely applied for diagnosis of malaria in most part of the endemic areas. But in case of submicroscopic and asymptomatic microscopic diagnosis is questioned. The study aims to develop a simple, cost effective & robust nucleic acid amplification technique for the detection of malaria parasite. METHODS: Study population included 50 clinically diagnosed positive malaria patient samples from various pathological laboratories. Microscopy by preparing thick film was carried out of every sample for primary screening in the available facility of Surat Raktadan Kendra & Research Centre- Blood Bank. The conventional PCR (Polymerase Chain Reaction) was applied for genus-specific amplification targeting the 18 S rRNA gene of Plasmodium. Agarose gel electrophoresis was used to separate and analyze the amplified PCR product using 2% Agarose gel. RESULTS AND CONCLUSION: The study shows that nested PCR not only detected all microscopic positive samples, but also detected submicroscopic infections that were missed or misread by microscopy. Hence, the sensitivity of molecular based detection technique is proved to be more compared to microscopic examination.

Mitochondrial genome provides species-specific targets for the rapid detection of early invasive populations of Hylurgus ligniperda in China.

BACKGROUND: Hylurgus ligniperda, a major international forestry quarantine pest, was recently found to have invaded and posed a serious threat to the Pinus forests of the Jiaodong Peninsula in China. Continuous monitoring and vigilance of the early population is imperative, and rapid molecular detection technology is urgently needed. We focused on developing a single-gene-based species-specific PCR (SS-PCR) method. RESULTS: We sequenced and assembled the mitochondrial genome of H. ligniperda to identify suitable target genes. We identified three closely related species for detecting the specificity of SS-PCR through phylogenetic analysis based on 13 protein-coding genes (PCGs). Subsequently, we analyzed the evolution of 13 PCGs and selected four mitochondrial genes to represent slow-evolving gene (COI) and faster-evolving genes (e.g. ND2, ND4, and ND5), respectively. We developed four species-specific primers targeting COI, ND2, ND4, and ND5 to rapidly identify H. ligniperda. The results showed that the four species-specific primers exhibited excellent specificity and sensitivity in the PCR assays, with consistent performance across a broader range of species. This method demonstrates the ability to identify beetles promptly, even during their larval stage. The entire detection process can be completed within 2-3 h. CONCLUSIONS: This method is suitable for large-scale species detection in laboratory settings. Moreover, the selection of target genes in the SS-PCR method is not affected by the evolutionary rate. SS-PCR can be widely implemented at port and forestry workstations, significantly enhancing early management strategies and quarantine measures against H. ligniperda. This approach will help prevent the spread of the pest and effectively preserve the resources of Chinese pine forests.

Digital PCR: A Tool to Authenticate Herbal Products and Spices.

Authentication of herbal products and spices is experiencing a resurgence using DNA-based molecular tools, mainly species-specific assays and DNA barcoding. However, poor DNA quality and quantity are the major demerits of conventional PCR and real-time quantitative PCR (qPCR), as herbal products and spices are highly enriched in secondary metabolites such as polyphenolic compounds. The third-generation digital PCR (dPCR) technology is a highly sensitive, accurate, and reliable method to detect target DNA molecules as it is less affected by PCR inhibiting secondary metabolites due to nanopartitions. Therefore, it can be certainly used for the detection of adulteration in herbal formulations. In dPCR, extracted DNA is subjected to get amplification in nanopartitions using target gene primers, the EvaGreen master mix, or fluorescently labeled targeted gene-specific probes. Here, we describe the detection of Carica papaya (CP) adulteration in Piper nigrum (PN) products using species-specific primers. We observed an increase in fluorescence signal as the concentration of target DNA increased in PN-CP blended formulations (mock controls). Using species-specific primers, we successfully demonstrated the use of dPCR in the authentication of medicinal botanicals.

Low-cost molecular methods to characterise gastrointestinal nematode co-infections of goats in Africa.

BACKGROUND: Veterinary diagnostics aid intervention strategies, track zoonoses, and direct selective breeding programs in livestock. In ruminants, gastrointestinal nematode (GIN) parasites are a major cause of production losses, but morphologically similar species limit our understanding of how specific GIN co-infections impact health in resource-limited settings. To estimate the presence and relative abundance of GINs and other helminths at the species level, we sought to develop a low-cost and low-resource molecular toolkit applied to goats from rural Malawi smallholdings. METHODS: Goats were subjected to health scoring and faecal sampling on smallholdings in Lilongwe district, Malawi. Infection intensities were estimated by faecal nematode egg counts with a faecal subsample desiccated for DNA analysis. Two DNA extraction methods were tested (low-resource magbead kit vs high-resource spin-column kit), with resulting DNA screened by endpoint polymerase chain reaction (PCR), semi-quantitative PCR, quantitative PCR (qPCR), high-resolution melt curve analysis (HRMC), and 'nemabiome' internal transcribed spacer 2 (ITS-2) amplicon sequencing. RESULTS: Both DNA isolation methods yielded comparable results despite poorer DNA purity and faecal contaminant carryover from the low-resource magbead method. GINs were detected in 100% of samples regardless of infection intensity. Co-infections with GINs and coccidia (Eimeria spp.) were present in most goats, with GIN populations dominated by Haemonchus contortus, Trichostrongylus colubriformis, Trichostrongylus axei, and Oesophagostomum columbianum. Both multiplex PCR and qPCR were highly predictive of GIN species proportions obtained using nemabiome amplicon sequencing; however, HRMC was less reliable than PCR in predicting the presence of particular species. CONCLUSIONS: These data represent the first 'nemabiome' sequencing of GINs from naturally infected smallholder goats in Africa and show the variable nature of GIN co-infections between individual animals. A similar level of granularity was detected by semi-quantitative PCR methods, which provided an accurate summary of species composition. Assessing GIN co-infections is therefore possible using cost-efficient low-resource DNA extraction and PCR approaches that can increase the capacity of molecular resources in areas where sequencing platforms are not available; and also open the door to affordable molecular GIN diagnostics. Given the diverse nature of infections in livestock and wildlife, these approaches have potential for disease surveillance in other areas.

Anaplasma and Ehrlichia Species in Ixodidae Ticks Collected from Two Regions of Bulgaria.

The aim of the study was to determine prevalence of Anaplasmataceae-infected ticks in the Black Sea Coast and the Pleven regions of Bulgaria. A total of 350 ticks from different tick species were collected. Two hundred fifty-five ticks were removed from dogs in the Black Sea Coast region, and 95 Ixodes ricinus ticks were collected by flagging vegetation with a white flannel cloth in two areas in the region of Pleven. After the DNA isolation of the ticks, a genus-specific polymerase chain reaction (PCR) was performed to identify Anaplasmataceae. Second PCRs were performed with species-specific primers to identify Ehrlichia canis (E. canis) and Anaplasma phagocytophilum (A. phagocytophilum). The results showed that 26.9% of the Ixodes ricinus ticks were infected with Anaplasmataceae in the Black Sea Coast region and 36.8% in the Pleven region. The infection with E. canis was detected in 35.7% and A. phagocytophilum in 25.0% of positive ticks from the Black Sea Coast region. In the Pleven region, 22.9% of ticks were positive for E. canis, while 42.9% were positive for A. phagocytophilum. The molecular identification of E. canis in ticks collected from Bulgaria was performed for the first time. In conclusion, the present study revealed a higher prevalence of ticks infected with Anaplasmataceae, particularly A. phagocytophilum, in the Pleven region than in the Black Sea Coast region.

Sporothrix pathogenic clade: Molecular analysis of animal and human clinical isolates.

Sporotrichosis is a subcutaneous mycosis that affects animals and humans. Varying in severity, occurrences range from local lesions to systemic involvement. It is caused by thermodimorphic and saprobic fungi from the Sporothrix pathogenic clade. This study aimed to identify the species and the sexual idiomorph distribution patterns responsible for diagnosed cases of sporotrichosis in São José do Rio Preto, Brazil. We included 188 isolates of Sporothrix sp. from feline lesions and 27 of human origin, which underwent molecular identification and genotyping for mating-type MAT1-1 and MAT1-2. The results showed that Sporothrix brasiliensis is the prevalent species in feline sporotrichosis outbreaks with the overwhelming presence of a single mating-type, MAT1-2 (P <.0001), suggesting a prevalently clonal form of spread. Morphological analyses did not discriminate among cryptic species in the genus Sporothrix, and molecular identification was essential for the correct identification of the species responsible for the observed cases of sporotrichosis. Distribution analyses of MAT1-2 isolates support the hypothesis of unidirectional migration from the current epidemics in São Paulo and Rio de Janeiro to the municipality of São José do Rio Preto.

This study aimed to identify the species and the sexual idiomorph distribution patterns responsible for diagnosed cases of sporotrichosis in São José do Rio Preto, Brazil. We included 188 isolates of Sporothrix sp. from feline lesions and 27 of human origin.

Comparing the VITEK 2 ANC card, species-specific PCR, and MALDI-TOF mass spectrometry methods for identification of lactic acid bacteria.

Lactic acid bacteria (LAB) are not only the most common probiotics in the food and feed industry but are also used as plant probiotics. Therefore, precise identification of LAB at the species level is required. In this study, we compared three different methods, the VITEK 2 ANC card, species-specific PCR, and MALDI-TOF MS, to identify six LAB (Lacticaseibacillus casei, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lactiplantibacillus plantarum, Lentilactobacillus buchneri, and Limosilactobacillus fermentum) species previously assigned to the genus Lactobacillus that are used as biofertilizers. Twenty-two strains of six LAB species were analyzed using the VITEK 2 ANC card, species-specific PCR, and MALDI-TOF MS, and identification rates at the species level were 45.5%, 95.5%, and 95.5%, respectively. There were cross-reactions between L. casei and L. parpacasei, and one strain of L. casei could not be identified by these three methods. PCR assays and MALDI-TOF MS were applicable for LAB identification. PRACTICAL APPLICATION: LAB are the most common probiotics in the food and feed industry, so precise identification and classification of LAB at the species level are required. This study aimed at comparing three different methods for the effective identification of six LAB species: biochemical testing using VITEK 2 ANC card, species-specific PCR, and MALDI-TOF MS analysis.

Development of allele-specific PCR methodology (AS-PCR) to screening A. lumbricoides and A. suum.

Ascaris lumbricoides and Ascaris suum are described as helminths that infect humans and pigs, respectively. It is estimated that infection by A. lumbricoides affects about 447 million individuals living in tropical regions of developing countries. However, there is an increasing number of cases of human ascariasis in countries with no recent history of autochthonous infection by A. lumbricoides. In these places, pigs have been incriminated as the main source of human infection. Conventional parasitological diagnosis does not allow species-specific identification, and the real epidemiological scenario of human and swine ascariasis is still uncertain. Therefore, this work presents the application of a species-specific molecular diagnosis, based on the allele-specific PCR methodology (AS-PCR), using the Internal Transcript Space 1 (ITS-1) of the ribosomal DNA, as a target for differentiating between the two species, using DNA obtained from eggs. To validate the methodology, stool samples positive for Ascaris spp, were obtained from 68 humans from seven Brazilian states and from six pigs from the state of Minas Gerais. All samples obtained from humans were genotyped as A. lumbricoides and all samples obtained from swine were genotyped as A. suum. These results are in agreement with the literature, which demonstrates that in most endemic regions, transmission cycles are separate. Therefore, the execution of this work allowed the availability of a useful methodology for the differential diagnosis of the species, which may contribute to the characterization of the real epidemiological profile of human and swine ascariasis, and to the implementation of future control strategies.

Report of autochthonous cases of localized cutaneous leishmaniasis caused by Leishmania (Leishmania) mexicana in vulnerable, susceptible areas of Southeastern Mexico

ABSTRACT Localized cutaneous leishmaniasis (LCL) is an endemic disease in several Mexican States with the main endemic areas located in the South-Southeast region of the country, where 90% of Leishmania (Leishmania) mexicana cases are registered. The Southeast region is located in the Yucatan Peninsula, including Campeche, Quintana Roo and Yucatan States. Campeche and Quintana Roo register more than 60% of the cases in the country each year, while in Yucatan the reports are of imported cases due to residents traveling to endemic areas. However, since 2015, autochthonous cases have been diagnosed by health authorities in municipalities with no previous transmission records. We aimed to identify Leishmania parasite species involved in autochthonous cases by means of the PCR technique. The present study included 13 autochthonous cases of LCL with clinical and parasitological diagnoses during 2018 and 2019 by health authorities, without specific identification of the causal agent. Tissue samples were taken by scraping the margins of active lesions and then they were spotted onto an FTATM Elute Microcard. Next, DNA was eluted and used for PCR amplification of specific Leishmania genus and L. (L.) mexicana species-specific fragments. Molecular analysis showed evidence that L. (L.) mexicana was the causal agent of LCL in 12 of the 13 patients; in one patient, PCR was not performed due to the patient's refusal to participate in the study. Identifying Leishmania species that cause LCL is necessary to define efficient treatment schemes and control strategies for the disease in vulnerable and susceptible areas of the Yucatan State's municipalities.

Ultra-fast PCR method for the distinguishing between Miichthys miiuy and Sciaenops ocellatus.

The mi-iuy croaker Miichthys miiuy has immense commercial value in the Republic of Korea. The red drum Sciaenops ocellatus is widely produced by aquaculture, although its price is approximately 25% that of M. miiuy. S. ocellatus has black spots on its tail, enabling it to be distinguished from M. miiuy based on appearance. However, identifying S. ocellatus after simple processing steps, such as skin removal and dicing, is difficult. Certain traders misrepresent and sell S. ocellatus as M. miiuy or cultured M. miiuy for illegal economical gain. Therefore, an accurate and rapid identification method is required to distinguish between M. miiuy and S. ocellatus in the field. Here, a method for rapid field identification was developed based on species-specific primers using a portable ultra-fast PCR instrument. The ultra-fast real-time PCR method can complete the entire analytical procedure, including DNA isolation, amplification, and detection, within 30 min, thus maintaining the accuracy of identifying M. miiuy and S. ocellatus products on site. Forty-nine commercial products were tested, and all samples were successfully identified. Thus, the developed method is rapid, efficient tool for ensuring consumer protection. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-021-00954-4.

Highly Clonal Structure and Abundance of One Haplotype Characterise the Diplodia sapinea Populations in Europe and Western Asia.

Diplodia sapinea is a cosmopolitan endophyte and opportunistic pathogen having occurred on several conifer species in Europe for at least 200 years. In Europe, disease outbreaks have increased on several Pinus spp. in the last few decades. In this study, the genetic structure of the European and western Asian D. sapinea population were investigated using 13 microsatellite markers. In total, 425 isolates from 15 countries were analysed. A high clonal fraction and low genetic distance between most subpopulations was found. One single haplotype dominates the European population, being represented by 45.3% of all isolates and found in nearly all investigated countries. Three genetically distinct subpopulations were found: Central/North European, Italian and Georgian. The recently detected subpopulations of D. sapinea in northern Europe (Estonia) share several haplotypes with the German subpopulation. The northern European subpopulations (Latvia, Estonia and Finland) show relatively high genetic diversity compared to those in central Europe suggesting either that the fungus has existed in the North in an asymptomatic/endophytic mode for a long time or that it has spread recently by multiple introductions. Considerable genetic diversity was found even among isolates of a single tree as 16 isolates from a single tree resulted in lower clonal fraction index than most subpopulations in Europe, which might reflect cryptic sexual proliferation. According to currently published allelic patterns, D. sapinea most likely originates from North America or from some unsampled population in Asia or central America. In order to enable the detection of endophytic or latent infections of planting stock by D. sapinea, new species-specific PCR primers (DiSapi-F and Diplo-R) were designed. During the search for Diplodia isolates across the world for species specific primer development, we identified D. africana in California, USA, and in the Canary Islands, which are the first records of this species in North America and in Spain.

Molecular detection of Phytophthora pluvialis, the causal agent of red needle cast in Pinus radiata.

BACKGROUND: Phytophthora pluvialis was first described in 2013 and is the causal agent of red needle cast (RNC) in Pinus radiata as well as infection in Douglas fir (Pseudotsuga menziesii). A species-specific PCR is necessary for detection of this pathogen and diagnosis of RNC. OBJECTIVE: To design and validate a species-specific molecular assay for P. pluvialis using isolates from infected pine needles. METHODS: Species-specific PCR primers were generated from the ras-related GTP-binding protein 1 gene (ypt1) gene sequence, concentrating on DNA regions unique to P. pluvialis, and real-time and quantitative polymerase chain reaction (qPCR) were used to detect P. pluvialis from both artificially inoculated and naturally infected samples. RESULTS: The species-specific PCR assay was generated following P. pluvialis DNA sequence analysis. In vitro tests of the specificity of the probe-based, quantitative, polymerase chain reaction (qPCR) assay showed that no amplification was observed with other Phytophthora species including other closely-related clade 3 species, or with fungal species associated with pine or with pine DNA. The limit of detection of the qPCR assay was 2 pg/µl. When the qPCR assay was used to detect P. pluvialis in artificially-inoculated and naturally infected P. radiata needles, a PCR product was detected in all inoculated samples; the mean concentration ranges of P. pluvialis DNA in the inoculated and naturally infected samples tested were 5.9-124.5 pg/µl and 8.1-340.2 pg/µl, respectively. The assays described herein were used with serological diagnostic strips, providing the ability to identify to species level. CONCLUSIONS: The assay described herein detects P. pluvialis with high specificity and sensitivity from a range of DNA samples, including those extracted from infected plant material and serological diagnostic strips. The ability to detect and identify P. pluvialis, from infected tissues directly, provides value and practicality to diagnostics, biosecurity and research.

Seasonal dynamics and life histories of three sympatric species of Pseudocalanus in two Svalbard fjords.

Small copepods are the most diverse and numerous group in high-latitude zooplankton, yet our knowledge of important species remains poor because of the difficulties involved in correct species identification. In this study, we use a molecular method of identification, a species-specific polymerase chain reaction, to provide the first description of the seasonal dynamics and life histories of the important genus Pseudocalanus in two Svalbard fjords with contrasting environments. We conducted monthly investigations in the relatively warm and ice-free Adventfjorden, supplemented with seasonal samples from the colder, seasonally ice-covered Billefjorden. We found three species of Pseudocalanus (the Arctic P. acuspes and P. minutus, and the boreal P. moultoni). Pseudocalanus acuspes had a distinct annual life cycle and dominated during summer, when it actively reproduced. Surprisingly, the boreal P. moultoni was present year-round in both fjords and was the dominant species during winter; the presence of all life stages of this species throughout the year suggests a more continuous reproduction. The Arctic P. minutus was the rarest of the three species and was likely able to complete its life cycle in Billefjorden but not in Adventfjorden. Our study demonstrates that closely related species may have different life strategies and environmental preferences, which presumably make high-latitude zooplankton communities more resilient to climate change impacts on genus but not necessarily on species level.

Blood meal analysis: host-feeding patterns of biting midges (Diptera, Ceratopogonidae, Culicoides Latreille) in Slovakia.

Biting midges of the genus Culicoides are vectors of important pathogens affecting domestic and wild animals and have played a major role in the re-emergence of new outbreaks of bluetongue (BTV) and Schmallenberg (SBV) viruses in Europe. To determine vector-host specificity, trophic preference from blood meal analysis is of major importance in the surveillance of arthropod-borne diseases. Of 28,752 specimens collected, we identified 17 Culicoides species and investigated a total of 48 host sequences from the blood meals. Culicoides obsoletus/C. scoticus, C. dewulfi, C. pulicaris, C. lupicaris, C. punctatus, C. newsteadi, C. riethi, and C. furcillatus were found to feed on mammals (cattle, horses, and humans), birds (domestic chickens), small rodents (Apodemus flavicollis), and hares (Lepus europaeus). To our knowledge, this is the first study investigating trophic preferences of Culicoides spp. in Slovakia. This study demonstrated that Culicoides species are able to feed on domesticated host vertebrates as well as birds, rodents, and humans.

TITLE: Analyse des repas sanguins : modes d'alimentation des hôtes des ceratopogonidés (Diptera, Ceratopogonidae, Culicoides Latreille) en Slovaquie. ABSTRACT: Les moucherons piqueurs du genre Culicoides sont des vecteurs d'agents pathogènes importants affectant les animaux domestiques et sauvages et ont joué un rôle majeur dans la réémergence de nouvelles épidémies de la fièvre catarrhale ovine (FCO) et de la maladie à virus Schmallenberg (SBV) en Europe. Pour déterminer la spécificité vecteur-hôte, la préférence trophique issue de l'analyse des repas sanguins est d'une importance majeure dans la surveillance des maladies transmises par les arthropodes. À partir de 28 752 spécimens collectés, nous avons identifié 17 espèces de Culicoides et étudié un total de 48 séquences d'hôtes à partir des repas de sang. Nous avons trouvé que Culicoides obsoletus / C. scoticus, C. dewulfi, C. pulicaris, C. lupicaris, C. punctatus, C. newsteadi, C. riethi et C. furcillatus se nourrissent à partir de mammifères (bovins, chevaux et humains), oiseaux (poulets domestiques), petits rongeurs (Apodemus flavicollis) et lièvres (Lepus europaeus). À notre connaissance, il s'agit de la première étude portant sur les préférences trophiques de Culicoides spp. en Slovaquie. Cette étude a démontré que des espèces de Culicoides sont capables de se nourrir d'hôtes vertébrés domestiqués ainsi que d'oiseaux, de rongeurs et d'humains.

Genetic diversity of Acidovorax oryzae in a single site in Mazandaran province, Iran.

Brown stripe, incited by Acidovorax oryzae is one of the most important and widespread diseases in rice (Oryza sativa) nurseries in Iran. Rice seedlings showing brown stripes were collected from suburb areas in southern Sari. Bacteria were isolated on plates of sorbitol neutral red agar. Species-specific PCR using trpB1/trpB2 and SEQID1/SEQID2 primer pairs resulted in amplification of the expected 478 bp and 514 bp long fragments, respectively. The 32 isolates subjected to REP-PCR analysis displayed 15 different banding profiles, with just one being shared by 10 isolates and 10 profiles were solitary and not shared by any isolates. Nonetheless, the isolates could not be different phenotypically. They appeared to be biochemically and nutritionally rather homogeneous while the genetic diversity as depicted in REP-PCR analysis was remarkable among the strains isolated from the closely located and even neighboring rice seedbeds. The implication of the findings in breeding programs is briefly discussed.

A New Duplex PCR-Assay for the Detection and Identification of Paracoccidioides Species.

Paracoccidioidomycosis (PCM) is a life-threatening systemic fungal infection caused by members of the Paracoccidioides brasiliensis complex and P. lutzii. Routine diagnoses of PCM down to the species level using classical mycological approaches are unspecific due to overlapping phenotypes. There is an urgent need for specific, sensitive, and cost-effective molecular tools to diagnose PCM. Variation among the exon-2 of the gp43 gene was exploited to design species-specific primer pairs to discriminate between members of the P. brasiliensis complex and P. lutzii in a duplex PCR assay. Primer-BLAST searches revealed highly species-specific primers, and no significant region of homology was found against DNA databases except for Paracoccidioides species. Primers PbraCx-F and PbraCx-R targeting P. brasiliensis DNA produced an amplicon of 308 bp, while primers Plu-F and Plu-R targeting P. lutzii DNA generated an amplicon of 142 bp. The lower limit of detection for our duplex PCR assay was 1 pg of gDNA. A panel of 62 Paracoccidioides revealed 100% specificity (AUC = 1.000, 95%CI 0.972-1.000, p < 0.0001) without cross-reacting with other medically relevant fungi or human DNA. As a proof of concept, we demonstrated the accurate identification of the P. brasiliensis complex (n = 7) or P. lutzii (n = 6) from a broad range of formalin-fixed, paraffin-embedded (FFPE) tissues of PCM patient's organs. In four cases, FFPE PCR results confirmed, for the first time, co-infection due to P. brasiliensis (S1) and P. lutzii in the same biopsy. Our duplex PCR assay is useful to detect and differentiate members of the P. brasiliensis complex and P. lutzii, providing clinical laboratories with an important tool to be applied routinely, especially in atypical cases such as those featuring negative serology and positive mycological examination of clinical specimens as well as for the investigation of putative co-infection cases. This will likely benefit thousands of infected patients every year in a wide area of the Americas.

Development of a species-specific PCR assay for authentication of Agkistrodon acutus based on mitochondrial cytochrome b gene

BACKGROUND: Agkistrodon acutus, a traditional Chinese medicine, clinically used in the treatment of rheumatism, tumor, and cardiovascular and cerebrovascular diseases. Due to the unique medicinal value and the difficulty of artificial breeding of Agkistrodon acutus, the supply of Agkistrodon acutus on the market exceeds the demand, and a large number of its adulterants are found on the market. In this study, the cytb gene sequences of Agkistrodon acutus and 9 snakes were compared and analyzed, specific primers were designed, and specific PCR methods were established to detect Agkistrodon acutus medicinal samples on the market. RESULTS: This method was successfully applied to distinguish the snake from other adulterated species, and tested 18 Agkistrodon acutus samples randomly purchased from six cities. Twelve samples were counterfeit and six were genuine. The standard reference material of Agkistrodon acutus was cloned by molecular cloning and sequencing, and the gene sequence difference with other species was significant. It shows that the region could be used as the fingerprint region of the target species. CONCLUSIONS: The proposed method can be used as a species-specific marker and can be highly distinguished from other adulterated snake species, which is helpful to effectively avoid the problem of false sale of Agkistrodon acutus.

Specific PCR-based detection of Phomopsis heveicola the cause of leaf blight of Coffee (Coffea arabica L.) in China.

Coffee (Coffea arabica L.) is currently grown in many tropical and subtropical areas countries and is a major traded commodity for the developing world. Coffee leaf blight, caused by Phomopsis heveicola, is one of the most important fungal diseases dangerous to coffee crops in China. This study aimed to develop a PCR-based diagnostic method for detecting P. heveicola in planta. Specific primers (CPHF/CPHR) were designed based on sequence data of region of internal transcribed spacer (ITS1 and ITS4) of P. heveicola. The efficiency and specificity of CPHF/CPHR were established by PCR analysis of DNA from P. heveicola strains isolated from China and fungal isolates of other genera. A single amplification product of 318 bp was detected from DNA P. heveicola isolates. No amplification product was observed with any of the other fungal isolates tested. The specific primers designed and employed in PCR detected P. heveicola up to 3 pg from DNA isolated. This is the first report on the development of a species-specific PCR assay for identification and detection of P. heveicola. Thus, the PCR-based assay developed was very specific, rapid and sensitive tool for the detection of pathogen P. heveicola.

Identification and Authentication of Commercial Mi-iuy Croaker (Miichthys miiuy) Products by Two PCR-Based Methods.

ABSTRACT: Mi-iuy croaker (Miichthys miiuy) is one of the most important ingredients of Korean cuisine and, thus, has a high economic value. However, the similar morphological traits among croaker fish belonging to family Sciaenidae are often exploited for seafood fraud. In this study, an M. miiuy-specific primer set was designed and further improved by the development of a rapid and cost-effective duplex PCR method. The specificity of M. miiuy-specific duplex PCR was tested using 22 seafood species, and no cross-reactivity was observed. The sensitivity of the PCR assay was found to be 0.1 ng/µL. For the first time, labeling compliance of 43 commercial mi-iuy croaker products was verified using both full DNA barcoding and M. miiuy-specific duplex PCR methods. For species identification, BOLDSYSTEMS and GenBank database were screened with the consensus sequences of each PCR product as a query. This identification result was further confirmed using the M. miiuy-specific duplex PCR method. The findings of this study revealed that principal species substituted were law croaker (Pseudotolithus senegallus, n = 4), bigeye croaker (Micropogonias megalops, n = 3), whitemouth croaker (Micropogonias furnieri, n = 1), and tigertoothed croaker (Otolithes ruber, n = 1). A significant percentage (21%) of mislabeling was present in commercial mi-iuy products sold on the South Korean market.