PCR-based Method For Detection Research Articles

Real-time PCR-based method for detection of Aspergillus Spp in bronchoalveolar respiratory samples

Real-time PCR-based method for detection of Aspergillus Spp in bronchoalveolar respiratory samples

A novel in vitro Caenorhabditis elegans transcription system

BackgroundCaenorhabditis elegans is an excellent model organism for biological research, but its contributions to biochemical elucidation of eukaryotic transcription mechanisms have been limited. One of the biggest obstacles for C. elegans biochemical studies is the high difficulty of obtaining functionally active nuclear extract due to its thick surrounding cuticle. A C. elegans in vitro transcription system was once developed by Lichtsteiner and Tjian in the 1990s, but it has not become widely used, most likely because the transcription reactions were re-constituted with nuclear extract from embryos, not from larval or adult worms, and the method of Dounce homogenization used to prepare the nuclear extract could lead to protein instability. Besides Dounce homogenization, several other techniques were developed to break worms, but no transcription reactions were re-constituted following worm disruption using these approaches. A C. elegans transcription system with effective preparation of functionally active nuclear extract from larval or adult worms has yet to be established. Additionally, non-radioactive methods for detecting transcription as alternatives to the conventional radioactive detection also need to be adapted into such an in vitro system.ResultsBy employing Balch homogenization, we achieved effective disruption of larval and adult worms and obtained functionally active nuclear extract through subcellular fractionation. In vitro transcription reactions were successfully re-constituted using such nuclear extract. Furthermore, a PCR-based non-radioactive detection method was adapted into our system to either qualitatively or quantitatively detect transcription. Using this system to assess how pathogen infection affects C. elegans transcription revealed that Pseudomonas aeruginosa infection changes transcription activity in a promoter- or gene-specific manner.ConclusionsIn this study, we developed an in vitro C. elegans transcription system that re-constitutes transcription reactions with nuclear extract of larval or adult worms and can both qualitatively and quantitatively detect transcription activity using non-radioactive approaches. This in vitro system is useful for biochemically studying C. elegans transcription mechanisms and gene expression regulation. The effective preparation of functionally active nuclear extract in our system fills a technical gap in biochemical studies of C. elegans and will expand the usefulness of this model organism in addressing many biological questions beyond transcription.

Revisiting museum collections in the genomic era: potential of MIG-seq for retrieving phylogenetic information from aged minute dry specimens of ants (Hymenoptera: Formicidae) and other small organisms

Multiplexed inter-simple sequence repeat genotyping by sequencing – MIG-seq – is an effective PCR-based method for genome-wide SNP detection using the Next-Generation Sequencing platform, and it provides a potential solution to a central problem in museomics – the difficulties of obtaining useful sequence data from aged specimens with often degraded and / or low yields of DNA. We demonstrate and validate the cost effectiveness and utility of the MIG-seq workflow in obtaining useful and robust sequence data from aged museum specimens. We applied the MIG-seq approach to 55 aged (10 - 23 years old) millimeter-sized dry-mounted specimens of the hyper-diverse ant genus Pheidole. A total of 50,782,736 reads were generated from the 55 samples (259,902 - 3,693,375 reads per sample). The reads corresponded to 36,862 SNPs from 4,849 polymorphic loci; the SNP dataset was then used to construct a Bayesian phylogenetic tree. The topology of the phylogenetic tree was highly compatible with existing knowledge of phylogenetic relationships among species of Pheidole. Therefore, we recommend the MIG-seq method as a cost-effective and highly applicable pipeline for conducting phylogenetic and population genetic studies on aged museum specimens, potentially enhancing the relevance of specimen repositories in general towards modern biodiversity science and conservation biology.Open access, licensed under CC BY 4.0. © 2020 The Author(s).DOI: 10.25849/myrmecol.news_030:151Pages: 151-159Volume: 30Year: 2020Journal: Myrmecol. News

Establishment of a Novel Molecular Detection Method for Sarcocystis in Venison.

Recently, horse meat (basashi) contaminated with Sarcocystis spp. caused food poisoning in Japan. An official detection method provided by the Ministry of Health, Labour and Welfare (MHLW), Japan, was designed to detect Sarcocystis fayeri to diagnose and control outbreaks of basashi food poisoning. In 2011, Sarcocystis-contaminated venison also caused food poisoning. However, the official MHLW detection method was not adequate for detecting Sarcocystis spp. in venison. In this study, we established a novel PCR-based detection method that amplifies 18S rRNA gene based on the conserved region of the sequence in 32 species of Sarcocystis for screening and quantification. Fifty venison samples from three areas in Hokkaido were examined by the MHLW method and the novel detection method. All samples were Sarcocystis spp.-positive. A sequence analysis indicated the presence of a species of Sarcocystis specific to sika deer (Cervus nippon), and not to horses. Another primer pair was designed for a quantitative real-time PCR assay to determine the copy number of the Sarcocystis-18S rRNA gene in parasitized venison. The melting curve analysis revealed high specificity of this assay. The calculated curve demonstrated that this quantitative PCR assay showed R2 value of 0.993 with 10-106 copies. Using this quantitative real-time PCR assay, the gene copy numbers were determined in 50 venison samples. The copy numbers of each sample ranged from 104 to 107 per gram. The copy numbers differed according to the area in Hokkaido. This indicates that the density of Sarcocystis spp. that infect Sika deer in Hokkaido is affected by the area. The novel screening and quantitative PCR method for Sarcocystis in venison was useful for collecting epidemiological information on Sarcocystis in wild Japanese sika deer, which will contribute to improve the safety of venison products in Japan.

A narrative review of single-nucleotide polymorphism detection methods and their application in studies of Staphylococcus aureus

Abstract Single-nucleotide polymorphisms (SNPs) are the third generation of genetic markers, having been refined from the first generation of restriction fragment length polymorphisms and the second generation of microsatellite polymorphisms. SNPs represent a focal point of current studies of Staphylococcus aureus. On one hand, this review aims to summarize common methodologies for detecting SNPs. These methods have typically included DNA genome sequencing methods and PCR-based detection methods. Alternative methods, such as mass spectrometry, denaturing high-performance liquid chromatography, SNaPshot, and SNP array have also been employed for SNP analysis. On the other hand, we enumerate a series of applications of SNP analysis in investigations of Staphylococcus aureus. SNP analysis can be applied to investigate epidemiological outbreaks and transmission of Staphylococcus aureus infections, the transmission and evolution of antimicrobial resistance genes in Staphylococcus aureus isolates, interactions of Staphylococcus aureus with other bacteria, and the links between Staphylococcus aureus in humans and livestock.

Optimized MALDI TOF Mass Spectrometry Identification of Francisella tularensis Subsp. holarctica.

Francisella tularensis is a tier 1 agent causing the zoonosis tularemia. This highly infectious Gram-negative bacterium is occasionally isolated from human samples (especially blood samples) in routine clinical microbiology laboratories. A rapid and accurate method for identifying this pathogen is needed in order to optimize the infected patient’s healthcare management and prevent contamination of the laboratory personnel. MALDI TOF mass spectrometry has become the gold standard for the rapid identification of most human pathogens. However, F. tularensis identification using such technology and commercially available databases is currently considered unreliable. Real-time PCR-based methods for rapid detection and accurate identification of F. tularensis are not available in many laboratories. As a national reference center for tularemia, we developed a MALDI TOF database allowing accurate identification of the species F. tularensis and its differentiation from the closely related neighbor species F. tularensis subsp. novicida and F. philomiragia. The sensitivity and specificity of this database were validated by testing 71 F. tularensis strains and 165 strains from 63 species not belonging to the Francisella genus. We obtained accurate identification at the species level and differentiation of all the tested bacterial strains. In particular, F. tularensis could be accurately differentiated from other small Gram-negative bacilli occasionally isolated from human samples, including species of the HACEK group and Brucella melitensis.

Loop-mediated isothermal amplification assay for pre-symptomatic stage detection of Xanthomonas axonopodis pv. punicae infection in pomegranate

Bacterial blight in pomegranate caused by Xanthomonas axonopodis pv. punicae (Xap) is an increasing threat to pomegranate cultivation in India. To prevent economic losses, it is pivotal to detect the infection in latent stages rather than in symptomatic stages. We have developed an enhanced loop-mediated isothermal amplification (LAMP) technique for the detection of latent Xap infection in pomegranate using six sets of pathovar-specific primers. The DNA-intercalating dye ethidium bromide was standardized for visualizing positive amplification in the LAMP assay. The optimum reaction time was 30 min or more at 65 °C, and assay sensitivity was enhanced down to femtogram (fg) amounts of template DNA (0.153 fg) in the LAMP assay. In artificially inoculated plants, the enhanced LAMP assay detected Xap on the 7th day post inoculation (dpi), while PCR detected Xap only after the 11th dpi. Finally, the specificity of the LAMP assay for Xap was validated against 31 bacterial strains belonging to 22 species. The LAMP assay developed in this study is highly specific, sensitive and robust and can be used as an improved alternative for PCR-based methods for the early detection of Xap infection in pomegranate. Early detection of Xap in pomegranate ensures quality fruits with increased yield by adopting precautionary measures that ensure improved monetary benefit to farmers.

Development of a Sensitive and Rapid Recombinase Polymerase Amplification Assay for Detection of Anaplasma phagocytophilum.

Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by the obligate intracellular Gram-negative bacterium Anaplasma phagocytophilum The disease often presents with nonspecific symptoms with negative serology during the acute phase. Direct pathogen detection is the best approach for early confirmatory diagnosis. Over the years, PCR-based molecular detection methods have been developed, but optimal sensitivity is not achieved by conventional PCR while real-time PCR requires expensive and sophisticated instruments. To improve the sensitivity and also develop an assay that can be used in resource-limited areas, an isothermal DNA amplification assay based on recombinase polymerase amplification (RPA) was developed. To do this, we identified a 171-bp DNA sequence within multiple paralogous copies of msp2 within the genome of A. phagocytophilum Our novel RPA assay targeting this sequence has an analytical limit of detection of one genome equivalent copy of A. phagocytophilum and can reliably detect 125 bacteria/ml in human blood. A high level of specificity was demonstrated by the absence of nonspecific amplification using genomic DNA from human or DNA from other closely-related pathogenic bacteria, such as Anaplasma platys, Ehrlichia chaffeensis, Orientia tsutsugamushi, and Rickettsia rickettsii, etc. When applied to patient DNA extracted from whole blood, this new RPA assay was able to detect 100% of previously diagnosed A. phagocytophilum cases. The sensitivity and rapidness of this assay represents a major improvement for early diagnosis of A. phagocytophilum in human patients and suggest a role for better surveillance in its reservoirs or vectors, especially in remote regions where resources are limited.

Utility of a PCR-based method for rapid and specific detection of toxigenic Microcystis spp. in farm ponds.

Microcystis is a widespread freshwater cyanobacterium that can produce microcystin, a potent hepatotoxin harmful to animals and humans. Therefore, it is crucial to monitor for the presence of toxigenic Microcystis spp. to provide early warning of potential microcystin contamination. Microscopy, which has been used traditionally to identify Microcystis spp., cannot differentiate toxigenic from non-toxigenic Microcystis. We developed a PCR-based method to detect toxigenic Microcystis spp. based on detection of the microcystin synthetase C (mcyC) gene and 16S rRNA gene. Specificity was validated against toxic and nontoxic M. aeruginosa strains, as well as 4 intergeneric freshwater cyanobacterial strains. Analytical sensitivity was as low as 747 fg/µL genomic DNA (or 3 cells/µL) for toxic M. aeruginosa. Furthermore, we tested 60 water samples from 4 farm ponds providing drinking water to swine facilities in the midwestern United States using this method. Although all water samples were positive for Microcystis spp. (i.e., 16S rRNA gene), toxigenic Microcystis spp. were detected in only 34 samples (57%). Seventeen water samples contained microcystin (0.1-9.1 μg/L) determined with liquid chromatography-mass spectrometry, of which 14 samples (82%) were positive for mcyC. A significant correlation was found between the presence of toxigenic Microcystis spp. and microcystin in water samples (p = 0.0004). Our PCR method can be a low-cost molecular tool for rapid and specific identification of toxigenic Microcystis spp. in farm ponds, improving detection of microcystin contamination, and ensuring water safety for farm animals.

Nationwide prevalence of Torque teno sus virus 1 and k2a in pig populations in Japan.

Because broad genetic diversity has recently been detected in Torque teno sus viruses (TTSuV1 and TTSuVk2), the viral genome detection method needs to be improved to understand the prevalence of these viruses. Here, we established single PCR-based detection methods for the TTSuV1 and TTSuVk2a genomes with newly designed primer pairs and applied them to investigate the prevalence of TTSuV1 and TTSuVk2a in Japanese pig populations. The results revealed that 98.2% and 81.7% of the pig farms tested positive for the TTSuV1 and TTSuVk2a genomes, respectively, indicating that both TTSuV1 and TTSuVk2a are widespread in Japan.

Development of TaqMan real-time RT-PCR for sensitive detection of diverse Raspberry ringspot virus isolates

Raspberry ringspot virus (RpRSV) is an important virus that infects horticultural crops including grapevine, cherry, berry fruit and rose. The genome sequences of RpRSV are highly diverse between isolates and this makes the design of a PCR-based detection method difficult. In this study, a TaqMan real-time RT-PCR assay was developed for the rapid and sensitive detection of RpRSV. Primers and probes targeting the most conserved region of the movement protein gene were designed to amplify a 229 bp fragment of RpRSV RNA-2. The assay was able to amplify all RpRSV isolates tested. The detection limit of the RpRSV target region was estimated to be 61–98 copies, depending on the RpRSV strain. The sensitivity was about 100 times greater than the conventional RT-PCR assay using the same primers as the real-time RT-PCR assay. A comparison with published conventional RT-PCR assays indicated that both published assays lacked reliability and sensitivity, as neither were able to amplify all RpRSV isolates tested, and both were at least 1000 times less sensitive than the novel TaqMan real-time RT-PCR assay. The assay can also be run as a duplex reaction with the nad5 plant internal control primers and probe to simultaneously verify the PCR competency of the samples. The amplicon obtained with the real-time RT-PCR assay is suitable for direct sequencing if it is necessary to further confirm the RpRSV identity or determine the RpRSV strain.

Detection and antibiotic resistance of Mycoplasma gallisepticum and Mycoplasma synoviae among chicken flocks in Egypt.

Background and Aim:Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the most significant pathogens of avian mycoplasmosis. This study aimed to isolate and identify MG and MS from chickens and detect the various virulence genes in the isolates. Moreover, the efficacies of different antibiotics were tested to identify suitable treatment regimens.Materials and Methods:We isolated MG and MS from 487 chicken samples of different ages located in different Governorates in Egypt using conventional isolation methods. The isolates were characterized by polymerase chain reaction (PCR) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then tested for antibiotic sensitivity by the minimum inhibitory concentration (MIC) method.Results:The prevalence of MG among the isolates was 9.85%, with the highest percentage isolated from air sacs, while the prevalence of MS among the isolates was 1.6%. Moreover, the highest levels of the prevalence of both MG and MS were during the winter and autumn sampling, while the lowest levels were in the summer and spring. Following the 16S rRNA-based detection of Mycoplasma isolates, 14 MG and 5 MS isolates were identified by different PCR-based detection methods for various virulence genes. Nine MG isolates contain the mgc2 gene, six MG isolates contain the gapA gene, and three MS isolates contain the vlhA gene. We validated a duplex PCR method for the simultaneous identification of MG and MS, based on 100% of the MG and MS isolates generating common bands at 55 and 17 kDa, respectively. The MIC method identified tiamulin and spiramycin as the antibiotics of choice for the treatment of MG and MS infections, respectively.Conclusion:For more precise diagnosis of Mycoplasma infections in chicken flocks, conventional isolation methods must be confirmed by PCR. SDS-PAGE analysis helps in epidemiological studies and vaccine preparation. The MIC method can be used to help develop therapies to control avian mycoplasmosis infections.

Viral Genome Sequencing for Ultrasensitive Detection of Circulating Tumor DNA

Accurate detection of circulating tumor DNA (ctDNA) has the potential to guide personalized cancer therapy. DNA viruses such as Epstein-Barr virus (EBV) and human papillomavirus (HPV) are major oncologic drivers of nasopharyngeal cancer (NPC) and cervical cancer (CC), respectively, and provide a convenient etiological marker for measurement of ctDNA. Polymerase chain reaction (PCR)-based methods have been widely studied, but more accurate tests are needed. We hypothesized that a next generation sequencing (NGS) approach could outperform PCR-based methods for viral ctDNA detection. We developed a novel NGS method for viral ctDNA detection. Viral genome sequencing for EBV (EBV-seq) or HPV (HPV-seq) was achieved by hybrid capture NGS. Unique molecular identifiers were incorporated for accurate copy number quantification. We implemented a custom bioinformatics workflow to (1) quantify viral sequences, (2) report viral subtype, and (3) measure ctDNA fragment length. The lower limit of detection (LLOD) was compared with published PCR-based methods in cell line dilution series. To test performance on clinical samples, we applied EBV-seq to a cohort of 19 newly diagnosed EBV-associated NPC patients for whom the clinical EBV quantitative PCR test had provided a false negative result and to a control cohort of 20 healthy volunteers. We applied HPV-seq to 40 plasma samples from a cohort of 18 CC patients treated with chemoradiotherapy and to a control cohort of 5 healthy volunteers. The LLOD of EBV-Seq was ≤0.01 EBV genome-equivalents (GE), 100X lower than quantitative PCR. The LLOD of HPV-seq was ≤0.2 HPV GE, 10X lower than digital PCR. For EBV-seq, 19/19 NPC patients had detectable ctDNA (sensitivity=100%, range=0.08-141 EBV GE); 10/20 controls had detectable ctDNA (specificity=50%, range=0.02-0.71 EBV GE). 17/19 (89%) patients, including 6/6 with stage I-II disease, had EBV-seq levels that were >20X above the highest level seen in the control cohort (i.e., 89% sensitivity with 100% specificity). For HPV-seq, 6/6 baseline CC samples, 10/17 (59%) end-of-treatment samples, 1/14 (7%) 3-month post-treatment samples, and 3/3 samples taken upon recurrence had positive HPV-seq results (sensitivity=100%, range=0.02-559 HPV GE) that were highly correlated with digital PCR (Pearson R=0.97) but with 60X higher signal than digital PCR; 0/5 controls had detectable ctDNA (specificity=100%). EBV-seq and HPV-seq were able to report viral subtype in 49/49 positive samples. Viral ctDNA was consistently shorter than non-cancer-derived plasma DNA (median difference=15bp). For DNA virus-associated cancers, viral genome NGS provides dramatically greater sensitivity than PCR-based methods and enables viral subtyping and ctDNA fragment length analysis. Further evaluation is warranted to test the clinical utility of ultrasensitive ctDNA detection using this approach.

Development of a new methodology for the detection of Colletotrichum truncatum and Fusarium sp. in bell pepper seed

This study was carried out towards the development of rapid PCR-based assays for detection and identification of Colletotrichum truncatum, C. gloeosporioides sensu lato and Fusarium sp. infecting green and red bell pepper seeds in Trinidad using DNA templates produced by automated grinding and robotic paramagnetic particle capture. DNA was extracted from freshly collected symptomatic green and red bell pepper seeds using Promega’s Maxwell® 16 robotic system. The PCR assay of 220 seeds harvested from field-infected bell pepper fruit compared different optimization parameters including seeds that were symptomatic and asymptomatic of infection, the relative success of two different DNA polymerase enzymes, genus-specific and species-specific primers that were pre-existing in the literature in addition to primers that were newly designed for this study and duplex PCR for the simultaneous detection of DNA templates belonging to two different fungal species. The findings indicated that seeds carried dual infections of C. truncatum and Fusarium sp. Compared to other primers that were screened, our newly designed primers, ITS219-F/ITS419-R with GoTaq® Green Master Hot Start Taq DNA Polymerase, detected C. truncatum in the highest number of symptomatic seeds of infected green and red bell pepper fruit as well as in asymptomatic seeds of infected red bell pepper fruit. Fusarium sp. was detected only in red bell pepper seeds. Duplex PCR using ITS219-F/ITS419-R and Fa/Ra primers successfully simultaneously detected and identified C. truncatum and Fusarium sp. in symptomatic seeds of infected red bell pepper fruit. None of the seeds were infected with C. gloeosporioides sensu lato which is currently considered to be a minor pathogen in bell pepper in Trinidad. PCR-based detection methods are key tools in the detection and identification of fungal plant pathogens especially where seeds are an important inoculum source. The molecular approach as optimized in this study, is faster, more sensitive and the presence of single or dual fungal infections can be assessed in one assay.

A High-Throughput Method as a Diagnostic Tool for HIV Detection in Patient-Specific Induced Pluripotent Stem Cells Generated by Different Reprogramming Methods.

Induced pluripotent stem cells (iPSCs) provide a unique opportunity for generation of patient-specific cells for use in translational purposes. We aimed to compare iPSCs generated by different reprogramming methods regarding their reprogramming efficiency, pluripotency capacity, and the possibility to use high-throughput PCR-based methods for detection of human pathogenic viruses. iPSCs from skin fibroblasts (FB), peripheral blood mononuclear cells (PBMCs), or mesenchymal stem cells (MSCs) were generated by using three different reprogramming systems including chromosomal integrating and nonintegrating methods. Reprogramming efficiencies were in accordance with the literature, indicating that the parental cell type and the reprogramming method play a major role for the reprogramming efficiencies (FB: STEMCCA: 1.30 ± 0.18, Sendai virus: 1.37 ± 0.01, and episomal plasmids: 0.04 ± 0.02; PBMCs: Sendai virus: 0.002 ± 0.001, episomal plasmids: 0) but result in the same characteristics of pluripotency. We found the highest reprogramming efficiencies for MSC with 3.32 ± 1.2 by using episomal plasmids. Since GMP standard working procedures and screening units need virus contamination-free cell lines, we studied HIV-1 contamination in the generated iPSCs. We used the high-throughput cobas® 6800/8800 system, which is normally used for detection of HIV-1 in plasma of patients, and found that footprint-free reprogramming methods as episomal plasmids and Sendai virus are useful for the described virus detection method. This fast, cost-effective, robust, and reliable assay demonstrates the feasibility to use high-throughput PCR-based methods for detection of human pathogenic viruses in ps-iPSC lines that were generated with nongenome integrating reprogramming methods.

Methods of isothermal nucleic acid amplification-based microfluidic chips for pathogen microorganism detection

Rapid detection of pathogenic microorganisms is key to the epidemiologic identification, prevention and control of disease in the field of public health. PCR-based pathogen detection methods have been widely used because they overcome the time-consuming issues that traditional culture-based methods required including the limited window required by immunological detection. However, the requirement on precision temperature-controlled thermal cyclers severely limits their use in resource-limited areas. The detection methods of pathogenic microorganisms based on isothermal amplification of nucleic acids are free of dependence on high-precision temperature control equipment, but requirements for nucleic acids extraction, amplification and detection must be defined. In recent years, a number of alternative methods for pathogenic microorganism detection have been developed by combining microfluidic technology with nucleic acid isothermal amplification technology. By designing the chip structures, optimizing the injection modes, and utilizing multiple detection and quantitative methods, the integration of pathogen nucleic acid extraction, amplification and detection is achieved. The method provides advantages of less instrument dependence, decreased operator requirements, smaller sample size, and higher automation which are suitable for the rapid detection of pathogenic microorganisms in various environments. In this review, we summarize several microfluidic detection methods based on nucleic acid isothermal amplification for pathogens including amplification principles, injection methods and detection methods. These methods provide more capability for the rapid screening of pathogenic microorganisms which enhances the management of infectious diseases in the field of public health.

Rapid detection of Burkholderia pseudomallei with a lateral flow recombinase polymerase amplification assay.

Melioidosis is a severe infectious disease caused by gram-negative, facultative intracellular pathogen Burkholderia pseudomallei (B. pseudomallei). Although cases are increasing reported from other parts of the world, it is an illness of tropical and subtropical climates primarily found in southeast Asia and northern Australia. Because of a 40% mortality rate, this life-threatening disease poses a public health risk in endemic area. Early detection of B. pseudomallei infection is vital for prognosis of a melioidosis patient. In this study, a novel isothermal recombinase polymerase amplification combined with lateral flow dipstick (LF-RPA) assay was established for rapid detection of B. pseudomallei. A set of primer-probe targeting orf2 gene within the putative type III secretion system (T3SS) cluster genes was generated and parameters for the LF-RPA assay were optimized. Result can be easy visualized in 30 minutes with the limit of detection (LOD) as low as 20 femtogram (fg) (ca. 25.6 copies) of B. pseudomallei genomic DNA without a specific equipment. The assay is highly specific as no cross amplification was observed with Burkholderia mallei, members of the Burkholderia cepacia-complex and 35 non-B. pseudomallei bacteria species. Moreover, isolates from patients in Hainan (N = 19), Guangdong (N = 1), Guangxi (N = 3) province of China as well as in Australia (N = 3) and Thailand (N = 1) were retrospectively confirmed by the newly developed method. LODs for B. pseudomallei-spiked soil and blood samples were 2.1×103 CFU/g and 4.2×103 CFU/ml respectively. The sensitivity of the LF-RPA assay was comparable to TaqMan Real-Time PCR (TaqMan PCR). In addition, the LF-RPA assay exhibited a better tolerance to inhibitors in blood than TaqMan PCR. Our results showed that the LF-RPA assay is an alternative to existing PCR-based methods for detection of B. pseudomallei with a potentiality of early accurate diagnosis of melioidosis at point of care or in-field use.

PCR-based Specific Detection of Bacillus in Liquid Organic Fertilizer

Rapid molecular PCR-based detection method for Bacillus species used in the production of Beyonic® liquid organic fertilizer was carried out based on nucleotide sequence data from the 16S rRNA gene. The method involved sequencing the 16S rRNA gene of several Bacillus species and identifying around 16-22 specific nucleotide bases from 5' and 3' ends in the Bacillus 16S rRNA gene sequences. One specific primer pair for Bacillus detection was determined as follow: 5' - CAT AAG ACT GGG ATA ACT CCG GG - 3' (forward) from positions of 85-107 bp, and 5’ - CCA GGC GGA GTG CTT AAT GC - 3’ (reverse) from positions of 836-854 bp. PCR assay and gel electrophoresis analysis showed that the primer pair was specific to the genus Bacillus.

Polyvalent detection of twelve viruses and four viroids affecting tomato by using a unique polyprobe

Non-radioactive molecular hybridization represents an attractive approach for the detection of multiple plant virus and/or viroids and a good alternative to the more extended serological and PCR-based detection methods. The use of polyprobes or riboprobes carrying partial sequences of different plant viruses or viroids fused in tandem, has permitted the detection of up to 10 different pathogens or the development of genus-specific probes. In the present article, the polyprobe technology has been adapted for the detection of the main viruses and viroids affecting tomato crops. To do this, three polyprobes have been developed covering four viroids (Poly4), twelve viruses (Poly12) or the four viroids plus the twelve viruses (poly16). The detection limit of the three polyprobes was comparable to the individual probes allowing the detection of up to 0,2 pg/μl of viral or viroidal RNA. A survey of 50 field samples revealed that all positive samples detected with the individual probes were also detected with the corresponding poly12 (98%) or poly16 (100%) probes. The analysis of tomato seeds revealed that both, single and polyprobes, were able to detect an infected seed in a pool of 250 healthy seeds. Finally, a ring-test analysis among six laboratories revealed a high reproducibility of the non-radioactive molecular hybridization procedure using the three polyprobes. The use of this technology in the routine analysis of tomato samples is discussed.

Rapid identification of Erwinia amylovora and Pseudomonas syringae species and characterization of E. amylovora streptomycin resistance using quantitative PCR assays.

Erwinia amylovora and Pseudomonas syringae are bacterial phytopathogens responsible for considerable yield losses in commercial pome fruit production. The pathogens, if left untreated, can compromise tree health and economically impact entire commercial fruit productions. Historically, the choice of effective control methods has been limited. The use of antibiotics was proposed as an effective control method. The identification of these pathogens and screening for the presence of antibiotic resistance is paramount in the adoption and implementation of disease control methods. Molecular tests have been developed and accepted for identification and characterization of these disease-causing organisms. We improved existing molecular tests by developing methods that are equal or superior in robustness for identifying E. amylovora or P. syringae while being faster to execute. In addition, the real-time PCR-based detection method for E. amylovora provided complementary information on the susceptibility or resistance to streptomycin of individual isolates. Finally, we describe a methodology and results that compare the aggressiveness of the different bacterial isolates on four apple cultivars. We show that bacterial isolates exhibit different behaviors when brought into contact with various apple varieties and that the hierarchical clustering of symptom severity indicates a population structure, suggesting a genetic basis for host cultivar specificity.