Targeting 16S rDNA Research Articles

Identification of diverse bacterial communities for potential bio-aids capable of troubleshooting for wastewater biological treatment processes

Three commercial bio-aid samples have shown good troubleshooting capabilities earlier during several biological wastewaters processing, but their microbial contents were still unclear. This study was aimed to assess eubacteria contents for the three bio-aids based on sequence comparisons of 16S ribosomal deoxyribonucleic acid (rDNA). Majority of target 16S rDNA sequences of bacteria within or cultured from bio-aids were obtained via pyrosequencing-based approach, and few sequences were from isolation- or cloning-sequencing approaches. Results showed that a total of 8114, 2327, and 5402 sequences were retrieved from pyrosequencing-based methods for the three bio-aids, respectively, which were affiliated with 100, 80, and 59 bacterial species based on sequence comparison using Ribosome Data Project (RDP) Classifier and Basic Local Alignment Search Tool (BLAST). Target sequences matched Bacillus sp., Enterococcus sp., and Clostridium sp. of Firmicutes phylum were the most abundant for the bio-aids, while some sequences identical to bacteria of phyla Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, and Proteobacteria were also found. Additionally, most of sequences from the isolation- or cloning-sequencing-based methods were not retrieved by the pyrosequencing-based methods. Culture media appeared to potentially select several bacteria species within phyla Firmicutes and/or Proteobacteria, but sequences from the culture-based methods did fulfill some absences for the uncultured method. Based on principal coordinate analysis, bacteria community structures of the three bio-aids were different. This study provided insights into bacteria contents for the three bio-aids as fundamental information in attempts for future selection or formulation of suitable bio-aids for solving various troubles during wastewater biological processing.

Detection and Characterization of the Emerging Relapsing Fever Pathogen, Borrelia miyamotoi, from the Ixodes ricinus Tick in the Rural Trakya (Thrace) Region of Northwestern Turkey.

The hard tick-borne relapsing fever agent, Borrelia miyamotoi infection in Ixodes ricinus ticks sampled from Istanbul and the countryside of Kirklareli in northwestern Turkey, was examined by TaqMan-PCR targeting 16S rDNA, nested PCR targeting 16S rDNA, the flagellin gene (flaB), and the 16S and 23S rDNA intergenic spacer (IGS), and sequencing analyses of these amplicons. B. miyamotoi was detected in 1 out of 248 I. ricinus ticks (infection rate 0.4%). The tick infected with B. miyamotoi was collected in Longos, Kirklareli province on the European side of Turkey near the Bulgarian border. The 16S rDNA, flaB, and IGS sequences from the infected tick showed high similarities to those of B. miyamotoi detected in I. ricinus in Europe.

Estimating sensitivity and specificity of a PCR for boot socks to detect Campylobacter in broiler primary production using Bayesian latent class analysis

The present study compares three different assays for sample collection and detection of Campylobacter spp. in broiler flocks, based on (i) the collection of faecal samples from intestinal organs (caecum), (ii) individual faecal droppings collected from the bedding and (iii) faecal material collected by socks placed on the outside of a pair of boots (boot socks) and used for walking around in the flock. The two first methods are examined for Campylobacter using a culture method (ISO-10272-2:2006), while the boot socks are tested using PCR. The PCR-assay is a genus specific multiplex PCR with primers targeting 16S rDNA in Campylobacter and primers targeting Yersinia ruckerii. Sixty-seven broiler flocks from Austria and 83 broiler flocks from Denmark were included in this prospective study and 89 of these were found to be positive in at least one method (AT: 49 samples, DK: 40 samples) whereas 61 of these were negative in all assays. In Austria samples for the three assays were collected simultaneously, which facilitates a direct comparison of the diagnostic test performance. In Denmark, however, boot socks and faecal droppings were collected three days before slaughter while caecum samples were collected at slaughter.The results were evaluated in the absence of a gold standard using a Bayesian latent class model. Austrian results showed higher sensitivity for PCR detection in sock samples (0.98; Bayesian credible interval (BCI) [0.93–1]) than for culture of faecal droppings (0.86; BCI [0.76–0.91]) or caecal samples (0.92; BCI [0.85–0.97]). The potential impact of Campylobacter introduction within the final three days before slaughter was observed in Denmark, where four flocks were tested negative three days before slaughter, but were detected positive at the slaughterhouse. Therefore the model results for the PCR sensitivity (0.88; BCI [0.83–0.97]) and cultural ISO-method in faecal samples (0.84; BCI [0.76–0.92]) are lower than for caecal samples (0.93; BCI [0.85–0.98]). In our study, PCR detection on boot sock samples is more sensitive than conventional culture. In view of the advantage of rapid results before slaughter and low costs for sampling, especially in combination with existing Salmonella surveillance systems (just another pair of boot socks needed), this method-matrix combination could be a valuable surveillance tool in the broiler primary production.

Rapid and quantitative detection of citrus huanglongbing bacterium ‘Candidatus Liberibacter asiaticus’ by real-time fluorescent loop-mediated isothermal amplification assay in China

Huanglongbing (HLB) is a devastating citrus disease that is associated with bacteria of the species ‘Candidatus Liberibacter asiaticus (Las)’ in China. All currently popular cultivars are susceptible to HLB, and there are no effective approaches to control the disease once the plants have been infected. Therefore, rapid and easy diagnostic tools are needed for maintaining clean nursery stock and inoculum removal strategies to control HLB. A real-time fluorescence loop-mediated isothermal amplification (RealAmp) targeting16S rDNA was developed for the rapid and quantitative detection of Las in China. The detection sensitivity of the RealAmp assay was approximately 1 pg/μl template DNA (equal to 2.4 × 105 target DNA copies/μl) and no cross-reaction was observed with other pathogens or virus-free seedlings. Besides the quantitative detection using SYTO-9 fluorescent dye, an improved closed-tube visual inspection technique using SYBR Green I staining was developed for rapid screening of samples and minimize the risk of cross-contamination. The RealAmp assay is an alternative quantitative method, which would be used as a routine detection service for nursery materials in China.

Phenotypic and genotypic approach to characterize Arcanobacterium pluranimalium isolated from bovine milk samples.

In the present study, three Arcanobacterium pluranimalium strains isolated from bovine milk samples of three cows of three farms (two cows with subclinical mastitis) could successfully be identified by phenotypical investigations, by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis and genotypically by sequencing the molecular targets 16S rDNA, 16S-23S rDNA intergenic spacer region (ISR), the β subunit of bacterial RNA polymerase encoding gene rpoB, the glyceraldehyde 3-phosphate dehydrogenase encoding gene gap, the elongation factor tu encoding gene tuf, and the pluranimaliumlysin encoding gene pla. The latter could also be identified by a loop-mediated isothermal amplification (LAMP) assay. The presented phenotypic and genotypic approaches might support the identification of A. pluranimalium in future and might help to understand the role this species plays in bovine mastitis.

Prevalence of Borrelia miyamotoi in Ixodes persulcatus in Irkutsk City and its neighboring territories, Russia

Adult Ixodes persulcatus were collected in highly populated districts in Irkutsk city, Russia, and in popular recreational and professional areas in its neighboring territories. Borrelia miyamotoi infection in I. persulcatus was examined using multiplex Taqman-PCR targeting 16S rDNA, and nested PCR and sequencing analyses targeting flaB and 16S rDNA. B. miyamotoi and Lyme disease Borrelia species were detected in 13 (infection rate, 2.9%) and 77 (17.3%) out of 445 I. persulcatus ticks, respectively, collected from 4 sites around the Baikal Lake. The 16S rDNA and flaB sequences of these amplicons were closely related to those of B. miyamotoi detected and/or isolated from I. persulcatus in Japan and Far Eastern Russia, and clustered separately from those of Europe and North America. These results indicate that additional surveillance for B. miyamotoi infection is needed in order to determine how it affects human health in Irkutsk City and its neighboring territories.

Food Targeting: A Real-Time PCR Assay Targeting 16S rDNA for Direct Quantification of Alicyclobacillus spp. Spores after Aptamer-Based Enrichment

Spore-forming Alicyclobacillus spp. are able to form metabolites that induce even in small amounts an antiseptical or medicinal off-flavor in fruit juices. Microbial contaminations could occur by endospores, which overcame the pasteurization process. The current detection method for Alicyclobacillus spp. can take up to 1 week because of microbiological enrichment. In a previous study, DNA aptamers were selected and characterized for an aptamer-driven rapid enrichment of Alicyclobacillus spp. spores from orange juice by magnetic separation. In the present work, a direct quantification assay for Alicyclobacillus spp. spores was developed to complete the two-step approach of enrichment and detection. After mechanical treatment of the spores, the isolated DNA was quantified in a real-time PCR-assay targeting 16S rDNA. The assay was evaluated by the performance requirements of the European Network of Genetically Modified Organisms Laboratories (ENGL). Hence, the presented method is applicable for direct spore detection from orange juice in connection with an enrichment step.

Evaluation of antimicrobial action of Carie Care™ and Papacarie Duo™ on Aggregatibacter actinomycetemcomitans a major periodontal pathogen using polymerase chain reaction.

Background:In the present scenario, we are made available with chemomechanical caries removal system containing a natural proteolytic enzyme for the ease in the excavation of infected dentine. The additive action for these agents is providing antimicrobial and anti-inflammatory properties.Aim:This study was undertaken for assessing the action of Carie Care™ and Papacarie Duo™ on Aggregatibacter actinomycetemcomitans.Materials and Methods:The samples were collected for cultivation of the periodontal pathogen from the clinical periodontal pockets using sterile paper points. The samples cultured under suitable conditions were analyzed with quantitative polymerase chain reaction targeting 16s r-DNA. The samples were divided into three groups namely, Group A: Control, Group B: With Papacarie Duo, Group C: With Carie Care. The pathogen inoculums plugs were inserted in the petri dishes containing chemically defined medium and the experimental gels at different concentrations and were incubated under optimal conditions. The inhibition of growth of the pathogen was studied visually.Results:There was visual inhibition of growth for Group B and C and also exhibited a dose-dependent effect also.Conclusion:Based on the results of the present study, Carie Care™ gel demonstrated better antimicrobial action against A. actinomycetemcomitans which is a major periodontal disease causing pathogen.

Molecular Analysis of Methanogen Richness in Landfill and Marshland Targeting 16S rDNA Sequences.

Methanogens, a key contributor in global carbon cycling, methane emission, and alternative energy production, generate methane gas via anaerobic digestion of organic matter. The methane emission potential depends upon methanogenic diversity and activity. Since they are anaerobes and difficult to isolate and culture, their diversity present in the landfill sites of Delhi and marshlands of Southern Assam, India, was analyzed using molecular techniques like 16S rDNA sequencing, DGGE, and qPCR. The sequencing results indicated the presence of methanogens belonging to the seventh order and also the order Methanomicrobiales in the Ghazipur and Bhalsawa landfill sites of Delhi. Sequences, related to the phyla Crenarchaeota (thermophilic) and Thaumarchaeota (mesophilic), were detected from marshland sites of Southern Assam, India. Jaccard analysis of DGGE gel using Gel2K showed three main clusters depending on the number and similarity of band patterns. The copy number analysis of hydrogenotrophic methanogens using qPCR indicates higher abundance in landfill sites of Delhi as compared to the marshlands of Southern Assam. The knowledge about “methanogenic archaea composition” and “abundance” in the contrasting ecosystems like “landfill” and “marshland” may reorient our understanding of the Archaea inhabitants. This study could shed light on the relationship between methane-dynamics and the global warming process.

Assessment of periodontal and opportunistic flora in patients with peri‐implantitis

To assess the presence of periodontal and opportunistic organisms in patients with peri-implantitis. Thirty-three partially edentulous subjects (22 women, 11 men), aged 32-90 years, who had one or more implants with peri-implantitis were included. Peri-implantitis was defined as: (i) the presence of bleeding on probing and/or suppuration and (ii) radiographic images showed marginal bone loss >1.8 mm after 1 year in function. Criteria for inclusion were: (i) partially edentulous patients having at least one implant diagnosed with peri-implantitis; (ii) no antibiotic therapy for 6 months prior to clinical examination. Following this definition, a total of 48 implants were diagnosed with peri-implantitis. Subgingival bacterial samples were obtained with sterile paper points from infected implants and selected teeth of each individual. Periodontopathogens (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia and Treponema denticola) were detected by multiplex PCR targeting 16S rDNA. Samples were placed in reduced transport medium and cultured for opportunistic pathogens (Staphylococcus aureus, enteric bacteria, Pseudomonas and yeasts). Twenty-two patients yielded positive results for P. gingivalis, 25 for T. forsythia, eight for P. intermedia and 13 for T. denticola. None of the patients yielded a positive result for A. actinomycetemcomitans. Non-periodontal species were found in five patients (15% of total). P. aeruginosa was found in four (12%) patients, and C. albicans (3%) and S. aureus in one patient (3%) each. In two cases of peri-implantitis, none of the periodontal or opportunistic microorganisms studied were detected in either implant or tooth samples. When results of the periodontopathic bacteria from the implant and tooth samples of the same patient were compared, 18 patients (54%) showed the same results for both samples and 15 (45%) patients different results. The implant surface may be colonized with pathogens different from periodontal bacteria. Opportunistic pathogens such as P. aeruginosa, S. aureus and C. albicans may be associated with implant failure.

Further Studies on Arcanobacterium phocisimile: a Novel Species of Genus Arcanobacterium.

Arcanobacterium phocisimile, a newly described species with the type strain A. phocisimile 2698T isolated from a vaginal swab of a harbour seal and four additional A. phocisimile strains also isolated from four harbour seals could reliably be identified by phenotypic properties, by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), and by sequencing the genomic targets 16S rDNA and 16S-23S rDNA intergenic spacer region and the genes rpoB and gap. The A. phocisimile strains investigated in the present study were isolated together with several other bacterial species indicating that the pathogenic importance of A. phocisimile remains unclear. However, the detection of peptidic spectra by MALDI-TOF MS and the presented phenotypic and genotypic approach might help to identify A. phocisimile in future.

Assessment of North Sea phytoplankton via molecular sensing: a method evaluation

Phytoplankton community analysis is important with respect to natural or humaninduced changes in the marine environment. Because of the efforts involved and the limitations of traditional methods, molecular sensing approaches are becoming more popular. Among others, microarray techniques targeting ribosomal 18S sequences have been successfully applied for phytoplankton investigation. In this contribution, we compared the results of two microarray methods targeting 18S rDNA and 18S rRNA with the results obtained from microscopy, HPLC and flow cytometry. On a qualitative basis, the microarrays showed similar or potentially better performance than the non-molecular methods. Quantitatively, our data suggest that microarray signals obtained from 18S rDNA provide relatively rough estimates of phytoplankton abundance. In contrast, when targeting 18S rRNA instead, a robust linear relationship (r2 ¼ 0.68) between molecular sensing signal and microscopic cell counts could be demonstrated using a probe specific to the genus Pseudo-nitzschia as an example. Thus, for both qualitative and quantitative purposes, microarray techniques can be valuable additions to traditional methods for phytoplankton analysis. Routine monitoring approaches in particular could benefit from advantages like reduced effort, higher taxonomic resolution and a potential for automation.

Development and evaluation of a TaqMan duplex real-time PCR quantification method for reliable enumeration of Candidatus Microthrix

Candidatus Microtrhix parvicella is one of the most common filamentous bacteria reported to be involved in bulking and foaming problems in activated sludge plants worldwide. In order to detect and quantify both M. parvicella and Microthrix calida by quantitative PCR (qPCR), primers targeting 16S rDNA genes were designed. The qPCR reaction was optimized by using the TaqMan technology and an internal positive control was included to ensure the absence of PCR inhibitors. A total of 29 samples originating from different wastewater treatment plants were analyzed and the results were compared by using conventional microscopy, fluorescent in situ hybridization and an existing SYBR Green-based assay. Our assay showed a 100% specificity for both M. parvicella and M. calida, a sensitivity of 2.93×109 to 29 copy numbers/reaction, an amplification efficiency of 93% and no PCR inhibition. By performing a spiking experiment including different Microthrix concentrations, recovery rates ranging from 65 to 98% were obtained. A positive correlation with the SYBR Green assay (R2=0.85) was found and most of the samples were in accordance with the microscopical observation. In comparison with SYBR Green assay, the probe-based TaqMan assay had a much lower detection limit. Compared with microscopy, some samples had a lower or higher enumeration when using qPCR. In conclusion, a qPCR method is forwarded here that could be useful as an early warning tool for fast and reliable detection of Microthrix in for instance sludge bulking events.

Nested polymerase chain reaction (PCR) targeting 16S rDNA for bacterial identification in empyema

Background:Empyema in children causes significant morbidity and mortality. However, identification of organisms is a major concern.Objective:To detect bacterial pathogens in pus specimens of children with empyema by 16S rDNA nested polymerase chain reaction (PCR) and correlate it with culture and sensitivity.Method:Sixty-six children admitted to the paediatric ward with a diagnosis of empyema were enrolled prospectively. Aspirated pus was subjected to cytochemical examination, culture and sensitivity, and nested PCR targeting 16S rDNA using a universal eubacterial primer.Results:Mean (SD) age was 5·8 (1·8) years (range 1–13). Analysis of aspirated pus demonstrated total leucocyte count >1000×106/L, elevated protein (≧20 g/L) and decreased glucose (≤2·2 mmol/L) in 80·3%, 98·5% and 100%, respectively. Gram-positive cocci were detected in 29 (43·9%) and Gram-negative bacilli in two patients. Nested PCR for the presence of bacterial pathogens was positive in 50·0%, compared with 36·3% for culture.Conclusion:16S rDNA PCR improves rates of detection of bacteria in pleural fluid, and can detect bacterial species in a single assay as well as identifying unusual and unexpected causal agents.

Further characteristics of Arcanobacterium canis, a novel species of genus Arcanobacterium

Comparable to previously conducted phenotypical and genotypical investigations characterizing Arcanobacterium canis, a newly described species with the type strain A. canis DSM 25104 isolated from an otitis externa of a dog, four additional A. canis strains isolated from infections of three dogs and one cat could reliably be identified by phenotypic properties, by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and by sequencing the genomic targets 16S rDNA, 16S-23S rDNA intergenic spacer region, 23S rDNA, and the genes rpoB and gap. All four A. canis investigated in the present study were isolated from the infected animals together with several other bacterial species indicating that the pathogenic importance of A. canis remains unclear. However, the detection of peptidic spectra by MALDI-TOF MS and the presented phenotypic and genotypic approaches might help to identify A. canis in future and might elucidate the role this species plays in infections of dogs and cats.

Use of PCR-DGGE Based Molecular Methods to Analyse Microbial Community Diversity and Stability during the Thermophilic Stages of an ATAD Wastewater Sludge Treatment Process as an Aid to Performance Monitoring.

PCR and PCR-DGGE techniques have been evaluated to monitor biodiversity indexes within an ATAD (autothermal thermophilic aerobic digestion) system treating domestic sludge for land spread, by examining microbial dynamics in response to elevated temperatures during treatment. The ATAD process utilises a thermophilic population to generate heat and operates at elevated pH due to degradation of sludge solids, thus allowing pasteurisation and stabilisation of the sludge. Genera-specific PCR revealed that Archaea, Eukarya and Fungi decline when the temperature reaches 59°C, while the bacterial lineage constitutes the dominant group at this stage. The bacterial community at the thermophilic stage, its similarity index to the feed material, and the species richness present were evaluated by PCR-DGGE. Parameters such as choice of molecular target (16S rDNA or rpoB genes), and electrophoresis condition, were optimised to maximise the resolution of the method for ATAD. Dynamic analysis of microbial communities was best observed utilising PCR-DGGE analysis of the V6-V8 region of 16S rDNA, while rpoB gene profiles were less informative. Unique thermophilic communities were shown to quickly adapt to process changes, and shown to be quite stable during the process. Such techniques may be used as a monitoring technique for process health and efficiency.

Molecular identification of dermatophytosis by polymerase chain reaction (PCR) and detection of source of infection by restricted fragment length polymorphism (RFLP)

Introduction Dermatophytes are responsible for most superficial fungal infections and the estimated lifetime risk of acquiring a dermatophyte infection is between 10-20%. These fungi are mainly classified in three major genera Microsporum, Trichophyton and Epidermophyton. Materials and Methods Clinically suspected 200 cases of dermatophyte infected patients from K. R. Hospital Mysore and Mission Hospital Mysore were included in this descriptive study from January 2011 to June 2012. All the culture positive smear- 10% Potassium Hydroxide (KOH) and culture in different dermatophytic medium patients were confirmed by PCR and source of infection was detected (n=10) from PCR positive patients and (n=10) from their domestic animals by PCR-RFLP methods targeting 18S rDNA regions of fungi. Results Out of 200 clinically suspected cases KOH mount was positive in 143 (71.5%) cases and culture was positive in 132(66%) cases. The isolates belonged to three genera and eight species as T.mentagrophytes 52(39.4%), T.rubrum 30(22.7%), T.violacium 18(13.6%), T.verrucosum 11(8.3%), E.floccosum 10(7.6%), M.canis 6(4.5%), T.tonsurans 03(2.3%) and T.schollenii 2(1.5%). To identify the source of infection 10 animals ,one each from the houses of 10 patients who were PCR positive were also subjected to PCR and RFLP. The animals and the patients were found to be infected by same organisms T.verrucosum .This indicates that T.verrucosum infection is from animal source. Conclusion Dermatophytic infections are more common infectious disease. Preliminary diagnosis of dermatophytosis can be done by KOH mount and culture, which takes longer time to report and cannot differentiate at the genus and species level. Results indicate that PCR-RFLP may be considered as gold standard for the diagnosis and confirmation of source of infection of dermatophytosis and can aid the clinician in initiating prompt and appropriate antifungal therapy. Journal of College of Medical Sciences-Nepal, 2012, Vol-8, No-4, 7-15 DOI: http://dx.doi.org/10.3126/jcmsn.v8i4.8694

Application of a qPCR Assay with Melting Curve Analysis for Detection and Differentiation of Protozoan Oocysts in Human Fecal Samples from Dominican Republic

A quantitative polymerase chain reaction assay with melt curve analysis (qPCR-MCA) was applied for the detection of protozoan oocysts in 501 human fecal samples collected in Dominican Republic. Samples were subjected to qPCR using universal coccidia primers targeting 18S rDNA to detect oocysts followed by MCA to identify oocyst species based on amplicon melting temperature. Putative positive samples were also tested by conventional PCR and microscopy. Cystoisospora belli (×3), Cryptosporidium parvum (×3), Cryptosporidium hominis (×5), Cryptosporidium meleagridis (×1), Cryptosporidium canis (×1), and Cyclospora cayetanensis (×9) were detected by qPCR-MCA and confirmed by sequencing. This assay consistently detected 10 copies of the cloned target fragment and can be considered more efficient and sensitive than microscopy flotation methods for detecting multiple species of oocysts in human feces. The qPCR-MCA is a reliable protozoan oocyst screening assay for use on clinical and environmental samples in public health, food safety and veterinary programs.

Humoral immune response and spreading of Encephalitozoon cuniculi infection in experimentally infected ponies

A total of 9 (8 stallions and 1 mare) 1 year old ponies were used for the experimental infection caused by Encephalitozoon cuniculi genotype II (107 spores per animal). Subsequently, individual horses were slaughtered 7, 14, 21, 28, 35, 42, 49, 56, and 63 days post infection. Immediately after slaughter, tissues samples of stomach, duodenum, jejunum, ileum, caecum, colon, spleen, liver, kidney, bladder, heart, lungs, and brain were sampled. In addition, urine, feces and blood specimens were collected. Enzyme-linked immunosorbent assay was used for determination of humoral immune response and nested PCR targeting 16S rDNA, whole ITS, and 5.8S rDNA was used for detection of E. cuniculi in collected organs, blood, feces and urine. No clinical signs of microsporidiosis including diarrhea or colic, neurological signs and fever were observed in any horses during whole experiment. Acute microsporidiosis in ponies was characterized by the dissemination of microsporidia into almost all organs and significant increase of concentration of specific antibodies in blood was observed from 28 to 42 DPI. After this acute stage microsporidia disappeared from most organs with the exception of the kidney, which was positive up to 63 DPI when the experiment was terminated. No pathological changes were observed in any organs with exception of one mare's brain, where E. cuniculi-positive cavity measuring 5cm×3cm in diameter formed in the lobus piriformis.

Development of a sensitive and specific LAMP PCR assay for detection of fish pathogen Lactococcus garvieae

Based on use of a loop-mediated isothermal amplification (LAMP) identification protocol, this study attempted to detect Lactococcus garvieae in fish by using primer sets designed from an L. garvieae alpha/beta fold family hydrolase gene. Reaction time and temperatures were optimized for 60 min at 60°C with the resulting amplicons visualized by adding SYBR Green I to the reaction tube. The assay specificity was assessed using 45 different bacterial strains. Positive results were observed in all 30 L. garvieae isolates from various aquatic animals. No false-positive results were observed in 15 non-L. garvieae strains. The detection limit of the LAMP assay was 10-fold more sensitive than the conventional polymerase chain reaction (PCR) targeting 16S rDNA when using purified L. garvieae DNA. The detection limit of the LAMP assay was approximately 300 colony-forming units (CFU) using crude bacterial lysates, 100-fold more sensitive than PCR. Furthermore, L. garvieae in spleen, kidney and brain of experimentally challenged tilapia and grey mullet were detected using this optimized LAMP assay. Results of this study demonstrate the effectiveness of LAMP in providing a rapid yet simple test for detecting L. garvieae in fish.