Species-specific PCR Detection Research Articles

Development of species-specific PCR detection for three Mycogone species causing wet bubble disease in white button mushroom

Agaricus bisporus, commonly known as white button mushroom, is a major agricultural crop worldwide. However, wet bubble disease in Agaricus bisporus, caused by three Mycogone species, namely Mycogone perniciosa, M. rosea and M. xinjiangensis, leads to tremendous economic losses in China. The three species are difficult to distinguish based on morphology and pathogenicity. An early, fast and accurate method to detect and differentiate among these species is essential for controlling the disease. Based on differences in Tef-1α gene sequences, therefore, three sets of species-specific primers, T-6-3/AT-4, T-2/AT-2 and T-7-1/AT-3, were successfully designed for the detection and identification of M. perniciosa, M. rosea and M. xinjiangensis, respectively, using an endpoint PCR assay. Subsequently, the specificities of the three primers were demonstrated using 43 isolates, including Mycogone spp., A. bisporus, Trichoderma harzianum, Trichothecium roseum, Cladobotryum dendroides, Lecanicillium fungicola, Fusarium solani and other soil-borne pathogens. The detection limits for M. perniciosa, M. rosea and M. xinjiangensis were 0.5 pg/μL, 0.2 ng/μL and 20 pg/μL of DNA template, respectively, suggesting strong amplification with the three primer pairs. Moreover, the species-specific primers efficiently detecteerd the target species in artificially inoculated mushrooms and soils. This is the first report to identify and distinguish each of the three Mycogone species. Besides, this PCR-based molecular method could provide a sensitive, quick and highly specific tool to discriminate among the three Mycogone species. Collectively, the assay developed in this study could have great application value for early detection of Mycogone species in soil and effective control of wet bubble disease.

Metabarcoding targeting the EF1 alpha region to assess Fusarium diversity on cereals.

Fusarium head blight (FHB) is a major cereal disease caused by a complex of Fusarium species. These species vary in importance depending on climatic conditions, agronomic factors or host genotype. In addition, Fusarium species can release toxic secondary metabolites. These mycotoxins constitute a significant food safety concern as they have health implications in both humans and animals. The Fusarium species involved in FHB differ in their pathogenicity, ability to produce mycotoxins, and fungicide sensitivity. Accurate and exhaustive identification of Fusarium species in planta is therefore of great importance. In this study, using a new set of primers targeting the EF1α gene, the diversity of Fusarium species on cereals was evaluated using Illumina high-throughput sequencing. The PCR amplification parameters and bioinformatic pipeline were optimized with mock and artificially infected grain communities and further tested on 65 field samples. Fusarium species were retrieved from mock communities and good reproducibility between different runs or PCR cycle numbers was be observed. The method enabled the detection of as few as one single Fusarium-infected grain in 10,000. Up to 17 different Fusarium species were detected in field samples of barley, durum and soft wheat harvested in France. This new set of primers enables the assessment of Fusarium diversity by high-throughput sequencing on cereal samples. It provides a more exhaustive picture of the Fusarium community than the currently used techniques based on isolation or species-specific PCR detection. This new experimental approach may be used to show changes in the composition of the Fusarium complex or to detect the emergence of new Fusarium species as far as the EF1α sequence of these species show a sufficient amount of polymorphism in the portion of sequence analyzed. Information on the distribution and prevalence of the different Fusarium species in a given geographical area, and in response to various environmental factors, is of great interest for managing the disease and predicting mycotoxin contamination risks.

Isolation, Identification and Genomic Analysis of Plesiomonas shigelloides Isolated from Diseased Percocypris pingi (Tchang, 1930)

Recently, the outbreak of a serious infectious disease of unknown etiology was noted in Percocypris pingi (Tchang, 1930) farms in Yunnan province. Due to currently limited information, we aimed to identify the pathogen isolates, determine the susceptibility of the isolates, evaluate the pathogenicity and analyze the genome of the representative strain. Ten strains of Gram-negative rods were isolated from diseased P. pingi and the isolates were identified as Plesiomonas shigelloides based on biochemical characteristics, 16S rRNA gene sequencing and species-specific PCR detection. The results of susceptibility analysis showed that two selected strains LS1 and LL2 were resistant to ampicillin, penicillin G, trimethoprim/sulfamethoxazole, tetracycline, enrofloxacin, nalidixic acid and enoxacin. A virulence assay indicated that the pathogen was virulent to zebrafish. Genomic analysis revealed that the LS1 isolate was closely related to strain GN7, which was isolated from animal farms. To the best of our knowledge, this is the first report of P. shigelloides as a pathogen of P. pingi. This study will provide a rational framework for exploration of epidemiological analysis of P. shigelloides in fish diseases and would further benefit conservation of the species.

Genus- and Species-Specific PCR Detection Methods.

PCR-based detection of fungal pathogens offers a sensitive and specific tool for the diagnosis of invasive fungal infections. A large variety of different clinical specimen types can be used as original material. However, certain precautions, in addition to the published MIQE guidelines [1], need to be taken to prevent contaminations from airborne fungal spores and PCR reagents. In addition, the European Aspergillus PCR Initiative (EAPCRI) recently defined standards for Aspergillus PCR [2, 3], following these recommendations leads to superior sensitivity. The combination of fungal PCR with the galactomannan ELISA assay increases the sensitivity for the detection of Aspergillus DNA from blood, compared to a single assay only [4, 5].

Simultaneous species-specific PCR detection and viability testing of poly(vinyl alcohol) cryogel-entrapped Rhodococcus spp. after their exposure to petroleum hydrocarbons

A method of simultaneous species-specific PCR detection and viability testing of poly(vinyl alcohol) cryogel-entrapped Rhodococcus spp. was developed that allowed the estimation of immobilized Rhodococcus opacus and Rhodococcus ruber survival after their exposure to petroleum hydrocarbon mixture. Spectrophotometric INT assay revealed high tolerance of gel-immobilized rhodococci to petroleum hydrocarbons, while among two Rhodococcus strains studied, R. ruber tolerated better to hydrocarbons compared to R. opacus. These findings were confirmed by respirometry results that showed increased respiratory activity of gel-immobilized Rhodococcus strains after 10-day incubation with 3% (v/v) petroleum hydrocarbon mixture. Moreover, jointly incubated rhodococcal strains demonstrated higher oxidative activities toward petroleum hydrocarbons than individual strains. Both Rhodococcus species were recovered successfully in cryogel granules using 16S rDNA-targeted PCR, even though the granules were previously stained with INT and extracted with ethanol. The method developed can be used for rapid detection and monitoring of gel-immobilized bacterial inocula in bioreactors or contaminated soil systems.

PCR-RFLP Identification of Prohibited Animal Derived DNA in Animal Feed

A method for confirming identification of prohibited species tissue in animal feed has been developed on the basis of PCR-RFLP analysis. In Japan, to prevent the spread of BSE through animal feed, the use of animal protein in feed has been regulated. Species-specific PCR detection of prohibited species materials in feed has been used as one of a series of laboratory tests to ensure the proper implementation of the feed regulations. However, since the result of this PCR method is determined only by amplicon length, it is sometimes necessary to confirm whether or not the positive result is due to the effect of a non-specific reaction. For this purpose, DNA sequencing is the best way to confirm the test result but it is not suitable for routine analysis because of the required time and cost. In this study, we developed an easy and rapid method to confirm the species identification (mammals, ruminants and cattle) by using 4 restriction enzymes: SmlI, MboI, BlnI and Hpy188III. This PCR-RFLP method, which ensures identification of prohibited animal species in feed, is useful for enhancing the reliability of feed inspection for BSE prevention. This method will be added to the Official Methods of Feed Analysis.

Morphologic, Genetic, and Biochemical Characterization of Helicobacter Magdeburgensis, a Novel Species Isolated from the Intestine of Laboratory Mice

The presence of enterohepatic Helicobacter species (EHS) is commonly noted in mouse colonies. These infections often remain unrecognized but can cause severe health complications or more subtle host immune perturbations and therefore can confound the results of animal experiments. The aim of this study was to isolate and characterize a putative novel EHS that has previously been detected by PCR screening of specific-pathogen-free mice. Biochemical analysis of enzyme activities (API campy), morphologic investigation (Gram-staining and electron microscopy) and genetic analyses (16SrRNA and 23SrRNA analyses, DNA fingerprinting, restriction fragment polymorphisms, and pulsed-field gel electrophoresis) were used to characterize isolated EHS. Genomic DNA fragments were sequenced to develop a species-specific PCR detection assay. Scanning electron microscopy revealed the presence of spiral-shaped EHS, which varied in length (2.5-6 μm) and contained single monopolar or single bipolar sheathed flagella. The bacteria were grown under anaerobic conditions, preferably on agar plates containing serum or blood. The 16SrRNA, genetic, and biochemical analyses indicated the identification of a novel EHS species, named Helicobacter magdeburgensis. We also examined the genome content using pulsed-field gel electrophoresis. Based on the pattern produced by two restriction enzymes, BamIII and KspI, the genome size was determined to be about 1.7-1.8 Mbp. We isolated and characterized a novel EHS species, H. magdeburgensis, morphologically, biochemically, and genetically. These results are important for future studies on the prevalence and pathophysiologic relevance of such infections. Our PCR assay can be used to detect and discriminate H. magdeburgensis from other Helicobacter species.

Species-specific PCR detection of the food-borne pathogen Vibrio parahaemolyticus using the irgB gene identified by comparative genomic analysis

Vibrio parahaemolyticus is an enteric pathogen, which can cause acute gastroenteritis in humans after consumption of raw or partially cooked seafood, and specific molecular markers are necessary for its accurate identification by PCR methods. In the present study, 23 protein-coding sequences were identified by the comparative genomics method as V. parahaemolyticus-specific candidate markers. We targeted the irgB gene (vp2603), coding for iron-regulated virulence regulatory protein IrgB, in order to develop a PCR method for the detection of V. parahaemolyticus. PCR specificity was identified by amplification of 293 V. parahaemolyticus templates and by the loss of a PCR product with 11 strains from other Vibrio species and 35 non-Vibrio bacterial strains. The PCR assay had the 369-bp fragment and the sensitivity of 0.17 pg purified genomic DNA from V. parahaemolyticus. Furthermore, a multiplex PCR assay for the detection of total and virulent strains of V. parahaemolyticus was developed by targeting irgB, tdh and trh genes. These data indicated that the irgB gene is a new and effective marker for the detection of V. parahaemolyticus. In addition, this study demonstrates that genome sequence comparison has a powerful application in identifying specific markers for the detection and identification of bacterial pathogens.

Stratified bacterial community in the bladder of the medicinal leech, Hirudo verbana

Most animals harbour symbiotic microorganisms inside their body, where intimate interactions occur between the partners. The medicinal leech, Hirudo verbana, possesses 17 pairs of excretory bladders that harbour a large number of intracellular and extracellular symbiotic bacteria. In this study, we characterized the bladder symbionts using molecular phylogenetic analyses, transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH). Restriction fragment length polymorphism (RFLP) and sequence analyses of 16S rRNA gene clone libraries suggested that six bacterial species co-colonize the leech bladders. Phylogenetic analyses revealed that these species belong to the alpha-Proteobacteria (Ochrobactrum symbiont), beta-Proteobacteria (Beta-1 and Beta-2 symbionts), delta-Proteobacteria (Bdellovibrio symbiont) and Bacteroidetes (Niabella and Sphingobacterium symbionts). Species-specific PCR detection and FISH confirmed the localization of the symbiotic bacteria in the bladders. The Ochrobactrum, Beta-1, Bdellovibrio and Sphingobacterium symbionts were consistently detected in 13 leeches from two populations, while infection rate of the other symbionts ranged between 20% and 100% in the two leech populations. Transmission electron microscopy observations of the bladders revealed epithelial cells harbouring a number of intracellular bacilli and an additional type of extracellular, rod-shaped bacteria in the luminal region. Fluorescence in situ hybridization with group-specific oligonucleotide probes revealed the spatial organization of the bacterial species in the bladder: the Ochrobactrum symbiont was located intracellularly inside epithelial cells; the Bacteroidetes were localized close to the epithelium in the lumen of the bladder; and the Bacteroidetes layer was covered with dense beta-proteobacterial cells. These results clearly demonstrate that a simple but organized microbial community exists in the bladder of the medicinal leech.

Species-specific PCR detection of the fish pathogen, Vibrio anguillarum, using the amiB gene, which encodes N-acetylmuramoyl-l-alanine amidase

Vibrio anguillarum is the causative agent of the fish disease vibriosis and is the most intensely studied species of Vibrio. In the present study, specific primers and a PCR assay were designed to detect V. anguillarum. The primers were designed to amplify a 429-bp internal region of the V. anguillarum amiB gene, which encodes the peptidoglycan hydrolase N-acetylmuramoyl-L-alanine amidase. PCR specificity was demonstrated by successful amplification of DNA from V. anguillarum and by the absence of a PCR product from 25 other Vibrio strains and various enteric bacteria. The PCR produced a 429-bp amplified fragment from as little as 1 pg of V. anguillarum DNA. The limit of detection for this PCR technique was c. 20 bacterial colonies in 25 mg of infected flounder tissue. These results suggest that this PCR system is a sensitive and species-specific detection method, and is possible to use as a diagnostic tool to detect V. anguillarum.

Direct and rapid detection by PCR of Erysipelothrix sp. DNAs prepared from bacterial strains and animal tissues.

A PCR method for rapid screening of Erysipelothrix spp. in the slaughterhouse was carried out by using four species-specific sets of oligonucleotide primers after initial amplification with the primer set MO101-MO102, which amplifies the 16S rRNA sequences of all four Erysipelothrix species. The DNA sequences coding for the rRNA gene cluster, including 16S rRNA, 23S rRNA, and the noncoding region downstream of 5S rRNA, were determined in order to design primers for the species-specific PCR detection system. The homology among the 4.5-kb DNA sequences of the rRNA genes of Erysipelothrix rhusiopathiae serovar 2 (DNA Data Bank of Japan accession no. AB019247), E. tonsillarum serovar 7 (accession no. AB019248), E. rhusiopathiae serovar 13 (accession no. AB019249), and E. rhusiopathiae serovar 18 (accession no. AB019250) ranged from 96.0 to 98.4%. The PCR amplifications were specific and were able to distinguish the DNAs from each of the four Erysipelothrix species. The results of PCR tests performed directly with tissue specimens from diseased animals were compared with the results of cultivation tests, and the PCR tests were completed within 5 h. The test with this species-specific system based on PCR amplification with the DNA sequences coding for the rRNA gene cluster was an accurate, easy-to-read screening method for rapid diagnosis of Erysipelothrix sp. infection in the slaughterhouse.