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Both Klebsiella pneumoniae and Chryseobacterium cause an increasing number of diseases in fish, resulting in great economic losses in aquaculture. In addition, the disease infected with Klebsiella pneumoniae or Chryseobacterium exhibited the similar clinical symptoms in aquatic animals. However, there is no effective means for the simultaneous detection of co-infection and discrimination them for these two pathogens. Here, we developed a duplex polymerase chain reaction (PCR) method based on the outer membrane protein A (ompA) gene of Klebsiella pneumoniae and Chryseobacterium. The specificity and validity of the designed primers were confirmed experimentally using simplex PCR. The expected amplicons for Klebsiella pneumoniae and Chryseobacterium had a size of 663 and 1404 bp, respectively. The optimal condition for duplex PCR were determined to encompass a primer concentration of 0.5 µM and annealing temperature of 57°C. This method was analytical specific with no amplification being observed from the genomic DNA of Escherichia coli, Vibrio harveyi, Pseudomonas plecoglossicida, Aeromonas hydrophila and Acinetobacter johnsonii. The limit of detection was estimated to be 20 fg of genomic DNA for Chryseobacterium and 200 fg for Klebsiella pneumoniae, or 100 colony-forming units (CFU) of bacterial cells in both cases. The duplex PCR was capable of simultaneously amplifying target fragments from genomic DNA extracted from the bacteria and fish liver. For practical validation of the method, 20 diseased fish were collected from farms, among which 4 samples were PCR-positive for Klebsiella pneumoniae and Chryseobacterium. The duplex PCR method developed here is time-saving, specific, convenient, and may prove to be an invaluable tool for molecular detection and epidemiological investigation of Klebsiella pneumoniae and Chryseobacterium in the field of aquaculture.
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Chryseobacterium , Enfermedades de los Peces , Klebsiella pneumoniae , Animales , Klebsiella pneumoniae/aislamiento & purificación , Klebsiella pneumoniae/genética , Chryseobacterium/aislamiento & purificación , Chryseobacterium/genética , Enfermedades de los Peces/microbiología , Enfermedades de los Peces/diagnóstico , Perciformes/microbiología , Infecciones por Klebsiella/veterinaria , Infecciones por Klebsiella/diagnóstico , Infecciones por Klebsiella/microbiología , Infecciones por Flavobacteriaceae/veterinaria , Infecciones por Flavobacteriaceae/microbiología , Infecciones por Flavobacteriaceae/diagnóstico , Acuicultura , ADN Bacteriano/aislamiento & purificación , ADN Bacteriano/genética , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y Especificidad , Reacción en Cadena de la Polimerasa Multiplex/veterinaria , Reacción en Cadena de la Polimerasa Multiplex/métodos , Cartilla de ADNRESUMEN
Huanglongbing (HLB) is a citrus infectious disease caused by 'Candidatus Liberibacter' spp. Recently, it has begun to spread rapidly worldwide, causing significant losses to the citrus industry. Early diagnosis of HLB relies on quantitative real-time PCR assays. However, the PCR inhibitors found in the nucleic acid extracted from plant materials pose challenges for PCR assays because they may result in false-negative results. Internal standard (IS) can be introduced to establish a single-tube duplex PCR for monitoring the influence of the PCR inhibitor, but it also brings the risk of false-negative results because the amplification of IS may compete with the target. To solve this problem, we proposed a mutation-enhanced single-tube duplex PCR (mSTD-PCR) containing IS with mutant-type primers. By introducing the 3'-terminal mutation in the primer of IS to weaken its amplification reaction and its inhibition of 'Candidatus Liberibacter asiaticus' (CLas) detection, the sensitivity and quantitative accuracy of CLas detection will not be affected by IS. In evaluating the sensitivity of CLas detection using simulation samples, the mSTD-PCR showed consistent sensitivity at 25 copies per test compared with the single-plex CLas assay. The detection result of 30 leaves and 30 root samples showed that the mSTD-PCR could recognize false-negative results caused by the PCR inhibitors and reduce workload by 48% compared with the single-plex CLas assay. Generally, the proposed mSTD-PCR provides a reliable, efficient, inhibitor-monitorable, quantitative screening method for accurately controlling HLB and a universal method for establishing a PCR assay for various pathogens.
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Citrus , Enfermedades de las Plantas , Reacción en Cadena en Tiempo Real de la Polimerasa , Rhizobiaceae , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Enfermedades de las Plantas/microbiología , Citrus/microbiología , Rhizobiaceae/genética , Rhizobiaceae/aislamiento & purificación , Cartilla de ADN/genética , Sensibilidad y Especificidad , Mutación , ADN Bacteriano/genética , Liberibacter/genéticaRESUMEN
Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the linearly single-stranded DNA viruses. Ecytonucleospora hepatopenaei (EHP) is an intracellular parasitic microsporidian. IHHNV and EHP are pathogens that have been widely prevalent in shrimp farming. Both of them are associated with growth retardation of the penaeid shrimp, which causes serious economic losses to shrimp farming. Shrimp can be co-infected with IHHNV and EHP. In this study, a rapid duplex polymerase chain reaction (PCR) was developed and optimized for the simultaneous detection of EHP and IHHNV. The detection limit of the duplex PCR could reach 1.5 × 102 copies for EHP and IHHNV. A total of 578 Litopenaeus vannamei samples were detected by the established duplex PCR detection method. The results suggested that 398 samples were infected with EHP, 362 samples were infected with IHHNV, and 265 samples were co-infected with EHP and IHHNV. The case-control analysis of the detected shrimp samples showed a certain synergistic effect between EHP and IHHNV.
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Densovirinae , Microsporidios , Penaeidae , Animales , Densovirinae/genética , Reacción en Cadena de la Polimerasa/métodos , Agricultura , Microsporidios/genéticaRESUMEN
Early and accurate detection of infectious diseases is a key step for surveillance, epidemiology and control, notably timely disease diagnosis, patient management and follow-up. In this study, we aimed to develop handheld ultra-fast duplex PCR assays coupled to amplicon detection by lateral flow (LF) immunoassay to deliver a rapid and simple molecular diagnostic test for concomitant detection and identification of the main Leishmania parasites encountered in Tunisia. We selected two DNA targets to amplify L. major/L. tropica and L. infantum/L. tropica groups of species DNAs, respectively. We optimized the experimental conditions of a duplex ultra-fast PCR. The amplification is performed using a portable Palm convection PCR machine within 18 min, and the products are detected using an LF cassette within 10 min. The test allows the identification of the infecting species according to the position and number of test lines revealed. Tested on a selection of DNAs of representative Leishmania strains of the three studied species (N = 37), the ultra-fast duplex PCR-LF showed consistent, stable and reproducible results. The analytical limit of detection of the test was 0.4 pg for L. major, 4 pg for L. infantum and 40 pg for L. tropica.
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Recently, studies have revealed that human herpesvirus 4 (HHV-4), also known as the Epstein-Barr virus, might be associated with the severity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Compared to SARS-CoV-2 infection alone, patients coinfected with SARS-CoV-2 and HHV-4 had higher risks of fever, inflammation, and even death, thus, confirming that HHV-4/SARS-CoV-2 coinfection in patients could benefit from clinical investigation. Although several intelligent devices can simultaneously discern multiple genes related to SARS-CoV-2, most operate via label-based detection, which restricts them from directly measuring the product. In this study, we developed a device that can replicate and detect SARS-CoV-2 and HHV-4 DNA. This device can conduct a duplex polymerase chain reaction (PCR) in a microfluidic channel and detect replicates in a non-labeled manner through a plasmonic-based sensor. Compared to traditional instruments, this device can reduce the required PCR time by 55% while yielding a similar amount of amplicon. Moreover, our device's limit of detection (LOD) reached 100 fg/mL, while prior non-labeled sensors for SARS-CoV-2 detection were in the range of ng/mL to pg/mL. Furthermore, the device can detect desired genes by extracting cells artificially infected with HHV-4/SARS-CoV-2. We expect that this device will be able to help verify HHV-4/SARS-CoV-2 coinfected patients and assist in the evaluation of practical treatment approaches.
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Early diagnosis of mucormycosis, a severe and potentially fatal complication in immunocompromised and COVID-19 patients, is crucial for initiating timely antifungal therapy and reducing infection mortality. In this study, the diagnostic performance of a duplex polymerase chain reaction (PCR) assay was evaluated to detect Mucorales-specific and Rhizopus oryzae-specific targets in 160 clinical samples collected from 112 COVID-19 patients suspected of invasive fungal rhinosinusitis (IFRS). During potassium hydroxide (KOH) direct microscopy, non-septate hyphae were observed in 73 out of 160 samples (45.63%); however, using duplex PCR, 82 out of 160 specimens (51.25%) tested positive. Among the positive PCR samples, 67 (81.71%) exhibited a double band (both 175 and 450 base pairs [bp]) indicating the presence of R. oryzae, and 15 (18.29%) showed only a single band (175 bp), suggesting the presence of non-R. oryzae Mucorales. DNAs from 10 microscopically negative samples and 4 samples with septate hyphae in microscopy were successfully amplified in PCR. Considering Calcofluor white fluorescence microscopy as the gold standard for laboratory diagnosis of mucormycosis, the duplex PCR assay utilized in this study exhibited a sensitivity of 93.88%, a specificity of 100%, a negative predictive value of 91.18%, and a positive predictive value of 100% for detecting mucormycosis in IFRS specimens. The duplex PCR assay demonstrated higher sensitivity compared to direct examination with KOH (82 vs. 73) and culture (82 vs. 41), enabling rapid detection/identification of Mucorales even in samples with negative culture or in biopsies with only a few hyphal elements.
Early diagnosis of mucormycosis, a severe complication in COVID-19 patients, is critical for reducing the mortality of the infection. In this study, a sensitive and rapid PCR assay to detect all Mucorales and delineate Rhizopus oryzae was developed and assessed to improve the diagnosis of mucormycosis.
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COVID-19 , Mucorales , Mucormicosis , Humanos , Mucormicosis/diagnóstico , Mucormicosis/veterinaria , COVID-19/diagnóstico , COVID-19/veterinaria , Mucorales/genética , Reacción en Cadena de la Polimerasa/veterinaria , Prueba de COVID-19/veterinariaRESUMEN
Norovirus (NoV) is a highly contagious enteric virus that causes widespread outbreaks and a substantial number of deaths across communities. As clinical surveillance is often insufficient, wastewater-based epidemiology (WBE) may provide novel pathways of tracking outbreaks. To utilise WBE, it is important to use accurate and sensitive methods for viral quantification. In this study, we developed a one-step duplex RT-qPCR assay to simultaneously test the two main human pathogenic NoV genogroups, GI and GII, in wastewater samples. The assay had low limits of detection (LOD), namely 0.52 genome copies (gc)/µl for NoVGI and 1.37 gc/µl for NoVGII. No significant concentration-dependent interactions were noted for both NoVGI and for NoVGII when the two targets were mixed at different concentrations in the samples. When tested on wastewater-derived RNA eluents, no significant difference between duplex and singleplex concentrations were found for either target. Low levels of inhibition (up to 32 %) were noted due to organic matter present in the wastewater extracts. From these results we argue that the duplex RT-qPCR assay developed enables the sensitive detection of both NoVGI and NoVGII in wastewater-derived RNA eluents, in a time and cost-effective way and may be used for surveillance to monitor public and environmental health.
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Norovirus , Humanos , Norovirus/genética , Aguas Residuales , Bioensayo , Brotes de Enfermedades , ARNRESUMEN
Hemoplasma species can cause infection varying from mild to severe in a wide range of hosts, including cattle and water buffalo. Two hemoplasma species, Mycoplasma wenyonii and Candidatus Mycoplasma haemobos, have been reported in cattle and water buffalo from different parts of the world to date. There was a lack of information on the presence and distribution of these pathogens in Turkey despite the negative economic impact on livestock production. This study aimed to develop a duplex PCR assay amplifying the 16S rRNA gene, in order to analyze DNA samples obtained from 297 cattle and 360 water buffaloes, and to determine the molecular prevalence of bovine hemoplasma species in Sivas province. Bovine hemoplasma species were found in 94 of 297 (31.64%) cattle and in 17 of 360 (4.72%) water buffaloes in this study. Randomly selected six positives PCR products (three samples each species) obtained from cattle and water buffaloes were sequenced, and the consensus sequences were uploaded to GenBank. Nucleotide similarity of 96.97-100% was determined between M. wenyonii isolates obtained in this study and those of M. wenyonii isolates present in the GenBank database, whereas C. Mycoplasma haemobos isolates from this study shared 99.04-100% homology with the C. Mycoplasma haemobos isolates uploaded to the GenBank. With the current study, the molecular presence of M. wenyonii and C. Mycoplasma haemobos were documented for the first time in cattle and water buffaloes in Turkey. Considering the rate of prevalence, veterinarians should take precautions against bovine hemoplasma species to protect animal health.
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Enfermedades de los Bovinos , Infecciones por Mycoplasma , Mycoplasma , Bovinos , Animales , Infecciones por Mycoplasma/epidemiología , Infecciones por Mycoplasma/veterinaria , Infecciones por Mycoplasma/diagnóstico , Búfalos , Prevalencia , ARN Ribosómico 16S/genética , Turquía/epidemiología , Enfermedades de los Bovinos/epidemiología , Mycoplasma/genéticaRESUMEN
This chapter presents a genotyping assay that uses DNA isolated from axenic cultures of Cryphonectria parasitica, which discriminates the six known diallelic vic loci and the two mating idiomorphs (MAT gene) based on (i) modified primer, labeled with a fluorescent dye, (ii) multiplex polymerase chain reaction (multiplex-PCR), and (iii) capillary electrophoresis system. Alternatively, we show that the same primer set is suitable for conventional PCR of each vic locus and MAT gene using nonmodified primer and agarose gel electrophoresis.
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Ascomicetos , Reacción en Cadena de la Polimerasa Multiplex , Ascomicetos/genética , Genotipo , Reproducción/genéticaRESUMEN
Theileriosis and anaplasmosis are important tick-borne hemoparasites of bovines. The first surveillance study aimed to assess the suitability of duplex PCR for simultaneous detection of Theileria annulata and Anaplasma marginale field infections in Jhang and Rawalpindi districts of Punjab, Pakistan. Cattle blood samples (n = 480) were collected from selected union councils of all tehsils using a multistage sampling technique. The sampling unit consisted of asymptomatic cattle belonging to either age, sex, and breed. Epidemiological data related to host, area, management, and season were collected using a questionnaire. Based on duplex PCR, the overall prevalence of the two concurrent tick-borne pathogens was 19.79% (95/480). Chi-square analysis indicated that age, breed, tick infestation, history of tick-borne diseases, frequency of acaricidial application, and season were significantly associated with tick-borne pathogens. Phylogenetic analysis of A. marginale and T. annulata isolates based on msp1ß and cytochrome b genes, respectively, revealed that nucleotide sequences acquired from these two pathogens are novel, grouped separately from different countries. All our A. marginale isolates showed 88.2 to 80.5% similarity with isolates from Egypt, Israel, Mexico, and lesser homology with South African isolates. Similarly, the phylogenetic tree based on cytochrome b partial sequences of T. annulata revealed that our sequences are closer to those from India and Iran. Based on this first study on concomitant detection of tick-borne pathogens, it can be concluded that mixed infections are endemic in the study districts and mPCR is suitable for detecting concurrent field infections. Simultaneous infections should be considered while performing surveillance and chemotherapeutic trials for better prevention and control of tick-borne diseases.
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Anaplasma marginale , Enfermedades de los Bovinos , Theileria annulata , Anaplasma marginale/genética , Animales , Bovinos , Enfermedades de los Bovinos/parasitología , Pakistán/epidemiología , Filogenia , Theileria annulata/genéticaRESUMEN
Anaplasma species are obligate intracellular rickettsial vector-borne pathogens that impose economic constraints on animal breeders and threaten human health. Anaplasma ovis and Anaplasma phagocytophilum infect sheep and goats worldwide. A duplex PCR targeting the msp2 and msp4 genes of A. phagocytophilum and A. ovis, respectively, was developed to analyze the field blood samples collected from sheep and goats. A total of 263 apparently healthy small ruminants from 16 randomly selected flocks situated in 3 bioclimatic zones in Tunisia were analyzed for Anaplasma infections. Anaplasma spp. was detected in 78.3% (95% confidence interval (CI): 72.8-83.1) of the analyzed animals. The prevalence of A. ovis in sheep (80.4%) and goats (70.3%) was higher than that of A. phagocytophilum (7.0% in sheep and 1.6% in goats). Using an inexpensive, specific, and rapid duplex PCR assay, we provide, to the best of our knowledge, the first molecular evidence for the presence of A. phagocytophilum in small ruminants in Tunisia. A. phagocytophilum generally presented as a co-infection with A. ovis. This study provides important data to understand the epidemiology of anaplasmosis in small ruminants, and highlights the risk of contracting the infection upon tick exposure.
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The loop-mediated isothermal amplification (LAMP) was standardized for rapid detection of Dichelobacter nodosus and Fusobacterium necrophorum. A total of 250 foot swabs were screened from sheep (200) and goats (50) from different districts of Rayalaseema, viz., Chittoor, Nellore, Kadapa, and Anantapur. Out of 250 samples 75 (30.0%) and 85 (34.0%) were positive for D. nodosus and F. necrophorum, respectively. All the 250 samples were screened individually for both the organisms by LAMP. Among them, 104 (41.6%) were found to be positive for D. nodosus and 120 (48.0%) were positive for F. necrophorum. The efficacy of LAMP in terms of sample DNA detection limit was compared with the PCR by using standard dilutions of DNA extracted from D. nodosus and F. necrophorum cultures. The detection limit was found to be higher than PCR for both the organisms. The sensitivity of LAMP is compared with PCR by targeting 16S rRNA gene of D. nodosus and lktA gene of F. necrophorum. In case of D. nodosus, out of 250 samples, 75 (30.0%) were positive by PCR and 104 (41.6%) were positive by LAMP. Among 250 samples, 85 (34.0%) were positive by PCR and 120 (48.0%) were positive by LAMP in case of F. necrophorum. The LAMP was found to be more sensitive than PCR in detecting the organisms with high statistical significance.
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Infecciones por Fusobacterium , Enfermedades de las Cabras , Infecciones por Bacterias Gramnegativas , Enfermedades de las Ovejas , Animales , Infecciones por Fusobacterium/veterinaria , Enfermedades de las Cabras/diagnóstico , Cabras , Infecciones por Bacterias Gramnegativas/veterinaria , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , ARN Ribosómico 16S/genética , Estándares de Referencia , Ovinos , Enfermedades de las Ovejas/diagnósticoRESUMEN
Nosemosis caused by Vairimorpha ceranae is one of the most important threats to honeybee colonies worldwide. This study aimed to determine the prevalence and intensity of Vairimorpha infection in different types of colonies and locations in Iran. In October 2017 and May 2018, 376 colonies from 97 apiaries were selected for each month according to a randomly clustered design. By considering 3-5 colonies for each apiary, 20 adult bees as pooled samples were collected from each colony. In microscopic analysis, 46.52% and 46.1% of samples in May and October showed Vairimorpha spores, respectively. The infection intensities in May and October were 5.94 ± 0.19 (× 106) and 5.86 ± 0.23 (× 106) spores/bee in a pooled sample, respectively. The mean infection intensity ranged from 1.8 to 12.5 (× 106) spores/bee. Statistically, there were no significant differences in the prevalence and intensity of V. ceranae infection between May and October samples. No significant differences were found among the prevalence rates of infection in the types of colonies; however, the intensity was significantly higher in migratory and mountainous colonies in May and only in migratory colonies in October. There was a significant correlation between the prevalence and intensity of V. ceranae infection (r2 = 0.695). PCR analysis showed that the samples were only infected with V. ceranae. No intraspecific variation to V. ceranae was found by direct sequencing of the amplified fragment of 16S rRNA. The obtained sequence was mainly 100% similar to those of V. ceranae isolates from European countries.
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Nosema , Animales , Abejas , Irán , Microsporidios , Nosema/genética , Filogenia , ARN Ribosómico 16SRESUMEN
Avian mycoplasmosis, mainly caused by Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS), is an economically important disease of poultry industry. The present study was aimed to develop duplex PCR as a rapid, specific and economical method for accurate detection of MG and MS in poultry and its comparison with single (monoplex) MG/MS PCR. During present investigation, a total of 146 poultry flocks having clinical history of respiratory disease were screened. Pooled tissue samples (trachea, lungs and air sacs) from 4-5 birds of each flock were collected during necropsy at disease investigation laboratories, Hisar, Haryana, India. The single and duplex PCR assays were standardized using primers of intergenic spacer region (IGSR; 16S-23S rRNA) for MG and hemagglutinin vlhA gene for MS, with expected amplicon size of 812 bp and 1200 bp products, respectively. In single PCR, 6.85%, 2.74% and 2.74% tissue samples were found positive for MG, MS and both MG and MS, respectively. However, duplex PCR showed, 7.53%, 2.74% and 1.37% positivity for MG, MS and both MG and MS, respectively. Taking the results of monoplex PCR as a gold standard, sensitivity and specificity of the developed duplex PCR was found to be 94.44% and 100%, respectively. Moreover, Cohen's kappa statistic (k = 0.97) measured a 'perfect' agreement between monoplex and duplex PCR assays. The positive and negative predictive values of duplex PCR was found to be 1.0 and 0.9922, respectively at 95% confidence interval (CI), as compared to monoplex PCR. The simultaneous use of two genes in a duplex PCR was more rapid and economical than two separate single PCR reactions.
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Infecciones por Mycoplasma/veterinaria , Mycoplasma gallisepticum/genética , Mycoplasma synoviae/genética , Enfermedades de las Aves de Corral/diagnóstico , Enfermedades de las Aves de Corral/microbiología , Animales , Pollos/microbiología , ADN Intergénico/genética , Infecciones por Mycoplasma/diagnóstico , Mycoplasma gallisepticum/aislamiento & purificación , Mycoplasma synoviae/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Aves de Corral/microbiología , ARN Ribosómico 16S/genética , ARN Ribosómico 23S/genética , Pavos/microbiologíaRESUMEN
Duplex polymerase chain reaction with lateral flow dipsticks (duplex PCR-LFD) was developed for the simultaneous detection of beta-lactamase Klebsiella pneumoniae carbapenemase (blaKPC ) and beta-lactamase New Dehli metallo-beta-lactamase (blaNDM ) genes in body fluid samples. This method was validated using well-characterized isolates. The assessment of the specificity of duplex PCR-LFD showed that there was no cross-reactivity with other targets. The detection limit of the duplex PCR-LFD assay was 20 CFU per ml for blaKPC and blaNDM . Among 177 sterile body fluid samples tested by the duplex PCR-LFD assay, 40 were blaKPC -positive and five were blaNDM -positive. The results obtained from 122 corresponding Gram-negative bacteria which were isolated from these clinical samples and tested by duplex PCR-LFD assay showed that there were 37 strains carrying blaKPC genes in 40 blaKPC -positive samples and three strains carrying blaNDM genes in five blaNDM -positive samples. Statistical analysis indicated that there was no significant difference between the direct detection of blaKPC and blaNDM genes in clinical sterile body fluid samples and their corresponding clinical isolates. Therefore, duplex PCR-LFD can be effective for the simultaneous detection of blaKPC and blaNDM in clinical isolates and directly from clinical samples, which may be helpful for the administration of appropriate antimicrobial treatment.
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Líquidos Corporales , beta-Lactamasas , Proteínas Bacterianas/genética , Bacterias Gramnegativas/genética , Humanos , Klebsiella pneumoniae/genética , Pruebas de Sensibilidad Microbiana , Técnicas de Amplificación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , beta-Lactamasas/genéticaRESUMEN
Meloidogyne incognita, the southern root-knot nematode (RKN), is the most predominant plant-parasitic nematode species of tomato and causes significant yield loss. The Mi-1.2 gene confers resistance in tomatoes to M. incognita; however, virulent RKN populations capable of parasitizing resistant tomato cultivars have been reported from different regions in the world. Four naturally occurring virulent populations of M. incognita were found in vegetable fields from four counties in Georgia with no history of tomato cultivation of the Mi gene. Two consecutive greenhouse trials showed that all four virulent RKN populations reproduced on tomato cultivars, including Amelia, Skyway, and Myrtle, with the Mi-1 gene, while an avirulent population of M. incognita race 3 was unable to overcome host resistance. Virulent RKN populations varied in reproduction among resistant cultivars, with Ma6 population having the greatest reproduction potential. No difference in penetration potential of the virulent (Ma6) and avirulent populations was found on susceptible and resistant tomato cultivars. However, virulent Ma6 population females were successful at egg-laying, whereas avirulent female development was arrested in the resistant cultivars. The virulent Ma6 population also induced feeding sites in the roots of resistant cultivars, whereas the avirulent population did not. To our knowledge, this is the first report of resistance-breaking populations of M. incognita in Georgia and the second state in the United States after California.
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Solanum lycopersicum , Tylenchoidea , Animales , Georgia , Solanum lycopersicum/genética , Tylenchoidea/genéticaRESUMEN
Sporothrix schenckii and allied species are thermodimorphic fungi widely distributed in nature which causes human and animal sporotrichosis, the most common subcutaneous mycosis globally. Sporotrichosis is acquired after a traumatic inoculation of soil or plant material contaminated with Sporothrix propagules or through bites and scratches from diseased cats. In Ascomycota, the master regulators of sex are MAT genes that lie in a single mating-type locus, in Sporothrix these are determined by two nonhomologous alleles, MAT1-1 and MAT1-2. We assessed the whole-genome sequences of medically relevant Sporothrix to develop a single-tube duplex PCR assay to screen S. brasiliensis, S. schenckii, S. globosa, and S. luriei idiomorphs (MAT1-1 or MAT1-2) and understand the distribution and incidence of mating-type strains from natural populations. Using our duplex PCR assay, a 673 bp amplicon (α-box protein) was consistently amplified from all MAT1-1 isolates, while a 291 bp fragment was only amplified from the isolates harboring MAT1-2 (HMG box). Molecular evidence suggests heterothallism (self-sterility) as the unique mating strategy among the species evaluated. The mating-type identity of 93 isolates revealed a nearly equal distribution (1:1 ratio) of mating type alleles within species but deviating between different outbreak areas. Remarkably, for S. brasiliensis in Rio de Janeiro, we report an overwhelming occurrence of MAT1-2 (1:13 ratio; χ2 = 10.286, P = 0.0013) opposing the high prevalence MAT1-1 in the Rio Grande do Sul (10:1 ratio; χ2 = 7.364, P = 0.0067). Therefore, the population structure of Sporothrix species refers from paucity to regular cycles of sexual recombination in most of the studied regions. Our PCR-based mating-type diagnostic assay is proposed here as an important marker to track the geographical expansion during the long-lasting outbreak of cat-transmitted sporotrichosis driven by S. brasiliensis.
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Sporothrix , Esporotricosis , Animales , Brasil , Reacción en Cadena de la Polimerasa , Sporothrix/genética , Esporotricosis/epidemiología , Esporotricosis/veterinariaRESUMEN
Coinfections with the tick-borne pathogens Theileria luwenshuni and Anaplasma phagocytophilum can cause significant economic losses in sheep and goat farming. The difficulty in detecting these two pathogens by microscopic examination warrants the development of a rapid detection test to discriminate them. In this study, a duplex polymerase chain reaction (PCR) assay was developed to simultaneously detect T. luwenshuni and A. phagocytophilum. Alignment of the sequences from related pathogens allowed us to design a primer pair targeting the 18S ribosomal RNA gene in T. luwenshuni and generate a target product of 962 bp, whereas a previously reported species-specific primer (SSAP2f/SSAP2r) for A. phagocytophilum was used in the same reaction to generate a product of 641 bp. Genomic DNA from T. luwenshuni and A. phagocytophilum was 10-fold serially diluted for testing PCR sensitivity. Under the optimal PCR conditions we established, the lower limit of detection of the assay was 29.13 fg/µL for T. luwenshuni and 1.53 fg/µL for A. phagocytophilum, and PCR primers used in this study were confirmed to be 100% species-specific using other hemoparasites previously identified by other methods. No significant difference was found between conventional and duplex PCR protocols used to detect the two species. Our study provides an effective, sensitive, specific, and accurate tool for the diagnosis and epidemiological surveillance of mixed infections of the two pathogens in sheep and goats.
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Anaplasma phagocytophilum , Enfermedades de las Cabras , Enfermedades de las Ovejas , Theileria , Anaplasma/genética , Anaplasma phagocytophilum/genética , Animales , Enfermedades de las Cabras/diagnóstico , Cabras , Reacción en Cadena de la Polimerasa/veterinaria , Sensibilidad y Especificidad , Ovinos , Enfermedades de las Ovejas/diagnóstico , Theileria/genéticaRESUMEN
Analyzing ambiguous bite marks using conventional morphological approaches to identify attackers is difficult; thus, applying molecular biological methods for identifying an attacker from their saliva is a possible approach in a forensic investigation. This study aimed to establish oral bacterial DNA-based human and canine saliva markers and develop a practical method for their discrimination. We considered Streptococcus oralis and Pasteurella canis as human and canine saliva marker candidates, respectively. Duplex bacterial DNA detection using melting curve analysis was designed and evaluated for forensic applicability using proof-of-concept experiments. S. oralis DNA was detected from human saliva samples from 30 out of 30 individuals, and P. canis DNA was detected from canine saliva samples from 73 out of 77 individuals (26 dog breeds). Additionally, both bacterial DNA markers were accurately detected from human blood-contaminated saliva samples and mock indistinct bite marks. Our results indicate that both bacterial DNA markers were sensitive, robust, and discriminating saliva markers. We consider that our duplex bacterial DNA examination is a simple, practical, and useful method for the detection of saliva from indistinct bite marks and discrimination between human and canine saliva.
Asunto(s)
Pasteurella , Saliva , Animales , ADN Bacteriano/genética , Perros , Marcadores Genéticos , HumanosRESUMEN
The accurate identification of Acinetobacter spp. is challenging due to their high phenotypic and biochemical similarities. Because clinical relevance and antibiotic susceptibility are significantly different among different genomic species of Acinetobacter, the exact identification of A. baumannii is necessary and it can help us prevent inappropriate antibiotic use and inferior clinical care. This project employed a sequence-specific PCR assay for the rpoB region in A. baumannii to distinguish it from non-Acinetobacter baumannii Acinetobacter species. Moreover, a duplex PCR assay was used to detect blaOXA-51-like and gluconolactonase genes as a second identification method. In this study, 210 isolates of Acinetobacter spp. were considered and identified by PCR-sequencing of rpoB gene as a reference test. PCR-sequencing of rpoB revealed that 179 isolates were A. baumannii and 31 were non- A. baumannii Acinetobacter strains. PCR amplification targeting the rpoB gene as the first method, detected 182 isolates of A. baumannii, while duplex PCR assay confirmed 163 isolates as A. baumannii. Data analysis indicated that the sensitivities of sequence-specific PCR of the rpoB gene and duplex PCR assay were 100% and 91.06%, respectively, while specificities were 91.18% and 100%, respectively. Given the data, it was revealed that these two methods showed a reasonable potential for the accurate identification of A. baumannnii from non- A. baumannii species. Sequence-specific PCR assay for the rpoB gene and duplex PCR assay for blaOXA-51-like and gluconolactonase genes are rapid, reliable and cost-effective methods which can be used in clinical laboratories for the accurate identification of A. baumannii.