Nested multiplex PCR—A feasible technique to study partial community of arbuscular mycorrhizal fungi in field-growing plant root

Plant can be infected by different arbuscular mycorrhizal fungi, but little is known about the interaction between them within root tissues mainly because different species cannot be distinguished on the basis of fungal structure. Accurate species identification of Arbuscular mycorrhizal fungi (AMF) colonized in plant roots is the comerstone of mycorrhizal study, yet this fundamental step is impossible through its morphological character alone. For accurate, rapid and inexpensive detection of partial mycorrhizal fungal community in plant roots, a nested multiplex polymerase chain reaction (PCR) was developed in this study. Five discriminating primers designed based on the variable region of the 5′ end of the large ribosomal subunit were used in the experiment for testing their specificity and the sensitivity in nested PCR by using spores from Glomus mosseae (BEG12), Glomus intraradices (BEG141), Scutellospora castaneae (BEG1) and two unidentified Glomus sp. HAUO3 and HAUO4. The feasibility assay of nested multiplex PCR was conducted by use of spore mixture, Astragalus sinicum roots co-inoculated with 4 species of arbuscular mycorrhizal fungi from pot cultures and 15 different field-growing plant roots respectively after analyses of the compatibility of primers. The result indicated that the sensitivity was in the same range as that of the corresponding single PCR reaction. Overall accuracy was 95%. The efficiency and sensitivity of this multiplex PCR procedure provided a rapid and easy way to simultaneously detect several of arbuscular mycorrhizal fungal species in a same plant root system.     

Nested multiplex PCR——A feasible technique to study partial community of arbuscular mycorrhizal fungi in field-growing plant root

Plant can be infected by different arbuscular mycorrhizal fungi, but little is known about the interaction between them within root tissues mainly because different species cannot be distinguished on the basis of fungal structure. Accurate species identification of Arbuscular mycorrhizal fungi (AMF) colonized in plant roots is the comerstone of mycorrhizal study, yet this fundamental step is impossible through its morphological character alone. For accurate, rapid and inexpensive detection of partial mycorrhizal fungal community in plant roots, a nested multiplex polymerase chain reaction (PCR) was developed in this study. Five discriminating primers designed based on the variable region of the 5′ end of the large ribosomal subunit were used in the experiment for testing their specificity and the sensitivity in nested PCR by using spores from Glomus mosseae (BEG12), Glomus intraradices (BEG141), Scutellospora castaneae (BEG1) and two unidentified Glomus sp. HAUO3 and HAUO4. The feasibility assay of nested multiplex PCR was conducted by use of spore mixture, Astragalus sinicum roots co-inoculated with 4 species of arbuscular mycorrhizal fungi from pot cultures and 15 different field-growing plant roots respectively after analyses of the compatibility of primers. The result indicated that the sensitivity was in the same range as that of the corresponding single PCR reaction. Overall accuracy was 95%. The efficiency and sensitivity of this multiplex PCR procedure provided a rapid and easy way to simultaneously detect several of arbuscular mycorrhizal fungal species in a same plant root system.     

Multiplex PCR assay for detection of Vibrio vulnificus biotype 2 and simultaneous discrimination of serovar E strains

In the present work we develop a multiplex PCR assay for the detection and identification of the fish pathogen Vibrio vulnificus biotype 2 with discriminating potential for zoonotic strains (serovar E). The PCR assay allowed the identification of two new biotype 2 serovar E human isolates from culture collections. Finally, the multiplex was successfully applied to both diagnosis and carrier detection in field samples.     

Multiplex PCR Assay for Detection of Vibrio vulnificus Biotype 2 and Simultaneous Discrimination of Serovar E Strains

In the present work we develop a multiplex PCR assay for the detection and identification of the fish pathogen Vibrio vulnificus biotype 2 with discriminating potential for zoonotic strains (serovar E). The PCR assay allowed the identification of two new biotype 2 serovar E human isolates from culture collections. Finally, the multiplex was successfully applied to both diagnosis and carrier detection in field samples.     

Reliable and rapid discrimination of congeneric species by mtDNA SNP analysis by multiplex PCR: application on three Trachurus and two Mullus fish species as model cases

We present a reliable, time-saving, and cost-effective multiplex PCR assay for discriminating congeneric species. Three fish species of the genus Trachurus and two of the genus Mullus served as model cases. Our multiplex PCR method interrogates species-specific diagnostic mitochondrial single nucleotide polymorphisms (mtSNPs). We selected two sets of mtSNPs that are organized in two multiplex systems for the Trachurus and the Mullus species, respectively. In both systems, all individuals tested could be safely assigned to one of the three Trachurus or two Mullus species. This novel SNP typing system offers a convenient and robust DNA profiling system suitable for large-scale identification of commercial fish species, for species with cryptic larvae or during juvenile stages, as well as for wildlife forensics. Handling editor: Christian Sturmbauer     

Multiplex PCR assay for rapid identification of three endangered snake species of India

Species identification has been the core issue in all approaches of conservation of endangered wild life. In this regard molecular techniques for species authentication have proved indispensable. A novel multiplex PCR assay for the identification of three Indian snake species Python morulus, Ptyas mucosus, and Naja naja is successfully demonstrated using 16S rRNA gene. Three reverse primers and a common forward primer were designed to generate three different size species-specific PCR fragments. Absence of any PCR amplification in non-target species proves the specificity of the primers. These four primers were combined in a multiplex assay to enable identification of three snake species in a single reaction. The assay described here shows its utility in identifying unknown snake specimen and in case of samples yielding low quality DNA. This multiplex PCR technique using novel primers is an unprecedented approach offered for forensic identification of exhibits originating from three Indian snake species. It is expected that this endeavor will help strengthening conservation efforts for these species.     

A species-specific real-time PCR assay for identification of three lichen-forming fungi, Lobaria pulmonaria, Lobaria immixta and Lobaria macaronesica

We present a multiplex real-time PCR assay for the simultaneous identification of three morphologically similar species of lichen-forming fungi, Lobaria pulmonaria, Lobaria immixta and Lobaria macaronesica. Based on TaqMan MGB (minor groove binding) probes targeting the fungal internal transcribed spacer (ITS nrDNA) region, our assay unambiguously identifies known samples from all the three species, thus providing a powerful alternative to the more expensive DNA-sequencing techniques.     

Comparison of PCR-RFLP with 21-plex PCR and rDNA Sequencing for Identification of Clinical Yeast Isolates

Non-albicans Candida species and other rare yeasts have emerged as major opportunistic pathogens in fungal infections. Identification of opportunistic yeasts in developing countries is mainly performed by phenotypic assay, which are time-consuming and prone to errors. The aim of the present study was to evaluate PCR-RFLP as a routinely used identification technique for the most clinically important Candida species in Iran and make a comparison with a novel multiplex PCR, called 21-plex PCR. One hundred and seventy-three yeast isolates from clinical sources were selected and identified with sequence analysis of the D1/D2 domains of rDNA (LSU rDNA) sequencing as the gold standard method. The results were compared with those obtained by PCR-RFLP using MspI restriction enzyme and the 21-plex PCR. PCR-RFLP correctly identified 93.4% of common pathogenic Candida species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and P. kudriavsevii (=?C. krusei)) and was able to identify 45.5% of isolates of the uncommon yeast species compared to the D1/D2 rDNA sequencing. Compared with PCR-RFLP, all common Candida species and 72.7% of uncommon yeast species were correctly identified by the 21-plex PCR. The application of the 21-plex PCR assay as a non-sequence-based molecular method for the identification of common and rare yeasts can reduce turnaround time and costs for the identification of clinically important yeasts and can be applied in resource-limited settings.

     

Multiplex PCR for rapid differentiation of three species in the "Clostridium clostridioforme group"

Clostridium clostridioforme is a relatively antimicrobial resistant, phenotypically heterogeneous anaerobe that has been involved in a variety of infections. 16S rDNA sequencing analysis revealed three principal species in what has been called Clostridium clostridioforme - Clostridium bolteae, C. clostridioforme, and Clostridium hathewayi. Based on the 16S rDNA sequence information we obtained, we developed a cost-effective, timesaving one-step multiplex PCR assay for rapid and accurate differentiation of these three species. The established multiplex PCR identification scheme was applied to the identification of 88 clinical isolates that had previously been identified phenotypically as C. clostridioforme. The identification obtained from multiplex PCR assays showed 100% agreement with 16S rDNA sequencing identification. This scheme will permit more accurate assessment of the role of these three Clostridium species in infection and of the degree of antimicrobial resistance in each of the species.     

A novel multiplex PCR assay for the identification of Indian crocodiles

Illegal hunting has been a major threat for the survival of wildlife fauna, including the three crocodile species that India harbours: Crocodylus palustris, Crocodylus porosus and Gavialis gangeticus. Although law prevents trade on these species, illicit hunting for trade continues to threaten the survival of these endangered species; conservation strategies therefore require a rapid molecular identification technique for Indian crocodiles. A multiplex polymerase chain reaction (PCR) assay with species-specific primers, considered as one of the most effective molecular techniques, is described herein. The primers were designed to yield species-specific sized amplicons. The assay discriminates the three Indian crocodile species unambiguously within a short time period using only simple agarose gel electrophoresis. We recommend this multiplex PCR assay to be used in the identification of Indian crocodile species.     

Rapid identification of the species of the Bacteroides fragilis group by multiplex PCR assays using group- and species-specific primers

We report a rapid and reliable two-step multiplex polymerase chain reaction (PCR) assay to identify the 10 Bacteroides fragilis group species - Bacteroides caccae, B. distasonis, B. eggerthii, B. fragilis, B. merdae, B. ovatus, B. stercoris, B. thetaiotaomicron, B. uniformis and B. vulgatus. These 10 species were first divided into three subgroups by multiplex PCR-G, followed by three multiplex PCR assays with three species-specific primer mixtures for identification to the species level. The primers were designed from nucleotide sequences of the 16S rRNA, the 16S-23S rRNA intergenic spacer region and part of the 23S rRNA gene. The established two-step multiplex PCR identification scheme was applied to the identification of 155 clinical isolates of the B. fragilis group that were previously identified to the species level by phenotypic tests. The new scheme was more accurate than phenotypic identification, which was accurate only 84.5% of the time. The multiplex PCR scheme established in this study is a simple, rapid and reliable method for the identification of the B. fragilis group species. This will permit more accurate assessment of the role of various B. fragilis group members in infections and of the degree of antimicrobial resistance in each of the group members.     

Identification of snails within the Bulinus africanus group from East Africa by multiplex SNaPshot trade mark analysis of single nucleotide polymorphisms within the cytochrome oxidase subunit I

Identification of populations of Bulinus nasutus and B. globosus from East Africa is unreliable using characters of the shell. In this paper, a molecular method of identification is presented for each species based on DNA sequence variation within the mitochondrial cytochrome oxidase subunit I (COI) as detected by a novel multiplexed SNaPshotTM assay. In total, snails from 7 localities from coastal Kenya were typed using this assay and variation within shell morphology was compared to reference material from Zanzibar. Four locations were found to contain B. nasutus and 2 locations were found to contain B. globosus. A mixed population containing both B. nasutus and B. globosus was found at Kinango. Morphometric variation between samples was considerable and UPGMA cluster analysis failed to differentiate species. The multiplex SNaPshotTM assay is an important development for more precise methods of identification of B. africanus group snails. The assay could be further broadened for identification of other snail intermediate host species.     

Multiplex PCR assay for the identification of nivalenol, 3- and 15-acetyl-deoxynivalenol chemotypes in Fusarium

The ability to rapidly distinguish trichothecene chemotypes in a given species/population of the genus Fusarium is important due to significant differences in the toxicity of these secondary metabolites. A multiplex PCR assay, based on primer pairs derived from the Tri3, Tri5 and Tri7 genes of the trichothecene gene cluster was established for the identification of the different chemotypes among Fusarium graminearum, F. culmorum and F. cerealis. Using the selected primers, specific amplification products of 625, 354 and 708 bp were obtained from Fusarium isolates producing nivalenol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol, respectively. Moreover, the multiplex PCR was successfully used to identify the chemotype of the Fusarium species contaminating wheat kernels. Four picograms of fungal DNA were found to be necessary to obtain a visible amplification product.     

Rapid molecular identification of Listeria species by use of real-time PCR and high-resolution melting analysis

Identification of Listeria species via a molecular method is critical for food safety and clinical diagnosis. In this study, an assay integrating real-time quantitative PCR (Q-PCR) with high-resolution melting (HRM) curve analysis was developed and assessed for rapid identification of six Listeria species. The ssrA gene, which encodes a transfer-messenger RNA (tmRNA) is conserved and common to all bacterial phyla, contains a variable domain in Listeria spp. Therefore, Q-PCR and a HRM profile were applied to characterize this gene. Fifty-three Listeria species and 45 non-Listeria species were detected using one primer set, with an accuracy of 100% in reference to conventional methods. There was a 93.3% correction rate to 30 artificially contaminated samples. Thus, Q-PCR with melting profiling analysis proved able to identify Listeria species accurately. Consequently, this study demonstrates that the assay we developed is a functional tool for rapidly identifying six Listeria species, and has the potential for discriminating novel species food safety and epidemiological research.     

Simultaneous detection and identification of trichothecene- and moniliformin-producing Fusarium species based on multiplex SNP analysis

AIM: To develop a multiplex identification method for trichothecene- and moniliformin-producing Fusarium species. METHOD AND RESULTS: In this article, we present a single nucleotide polymorphism (SNP) assay to simultaneously detect and identify 16 trichothecene- and moniliformin-producing Fusarium species. A number of SNP primers are designed to detect clades of species with particular mycotoxigenic synthetic abilities. The assay is based on minisequencing using SNaPshot reactions and the SNP primers are designed based on motifs derived from phylogenetic analyses of translation elongation factor-1alpha sequences. The present version of the Fusarium SNP assay can distinguish major groups of trichothecene producers; the strict-type-A, the strict-type-B, the type-A and type-B trichothecene producers and the putative moniliformin producers. The SNP assay was validated against five naturally infected cereal samples that previously had been analysed morphologically, chemically and by a multiplex DNA array hybridization. CONCLUSIONS: The Fusarium SNP assay reveals the advantages of using SNPs for multiplex species identification. Significance AND IMPACT OF THE STUDY: The current assay may qualify as a high-throughput screening method for small-grain cereals in the feed and food chain, and may facilitate detection of new or introduced Fusarium species.     

Specific PCR-primers as a reliable tool for the detection of white truffles in mycorrhizal roots

During their symbiotic phase, white truffles are barely distinguishable morphologically, and molecular probes are needed for their identification. Here we report the design of species-specific primers for two white truffles ( Tuber magnatum and T. borchii ) on the basis of their ITS sequence. Their efficiency has been successfully tested on fruit bodies of the same species, many related and unrelated fungal species, as well as mycorrhizal roots in direct, nested and multiplex PCR experiments. They have allowed us to identify T. magnatum mycorrhizas for the first time.     

Current Status in Diagnosis of Scedosporium Infections: What Is the Impact of New Molecular Methods?

Scedosporium species are increasingly encountered as fungal pathogens. Species identification is important due to species-specific differences in epidemiology, antifungal susceptibility and virulence. Histology and culture-based identification are hampered by their low sensitivity and specificity. The use of new selective media has improved the recovery rate from clinical samples. Molecular methods, including multiplex PCR, PCR-RFLP analysis, DNA sequencing, oligonucleotide arrays, real-time PCR, rolling circle amplification, are increasingly used for species identification. Most recently, Matrix-Assisted Laser Desorption-Time of Flight Mass Spectrometry has been successfully applied as a tool for rapid identification of clinically relevant Scedosporium species. This review aims to summarize the methods currently used to guide the clinical microbiology laboratory in the selection of the most appropriate identification techniques. This will aid the laboratory in making a fast and reliable diagnosis that enables the clinician to make correct treatment choices.     

SNaPAfu: A Novel Single Nucleotide Polymorphism Multiplex Assay for Aspergillus fumigatus Direct Detection,Identification and Genotyping in Clinical Specimens

Objective

Early diagnosis of invasive aspergillosis is essential for positive patient outcome. Likewise genotyping of fungal isolates is desirable for outbreak control in clinical setting. We designed a molecular assay that combines detection, identification, and genotyping of Aspergillus fumigatus in a single reaction.

Methods

To this aim we combined 20 markers in a multiplex reaction and the results were seen following mini-sequencing readings. Pure culture extracts were firstly tested. Thereafter, Aspergillus-DNA samples obtained from clinical specimens of patients with possible, probable, or proven aspergillosis according to European Organization for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria.

Results

A new set of designed primers allowed multilocus sequence typing (MLST) gene amplification in a single multiplex reaction. The newly proposed SNaPAfu assay had a specificity of 100%, a sensitivity of 89% and detection limit of 1 ITS copy/mL (∼0.5 fg genomic Aspergillus-DNA/mL). The marker A49_F was detected in 89% of clinical samples. The SNaPAfu assay was accurately performed on clinical specimens using only 1% of DNA extract (total volume 50 µL) from 1 mL of used bronchoalveolar lavage.

Conclusions

The first highly sensitive and specific, time- and cost-economic multiplex assay was implemented that allows detection, identification, and genotyping of A. fumigatus strains in a single amplification followed by mini-sequencing reaction. The new test is suitable to clinical routine and will improve patient management.     

Cryptic species within Anopheles longipalpis from southern Africa and phylogenetic comparison with members of the An. funestus group

House-resting Anopheles mosquitoes are targeted for vector control interventions; however, without proper species identification, the importance of these Anopheles to malaria transmission is unknown. Anopheles longipalpis, a non-vector species, has been found in significant numbers resting indoors in houses in southern Zambia, potentially impacting on the utilization of scarce resources for vector control. The identification of An. longipalpis is currently based on classical morphology using minor characteristics in the adult stage and major ones in the larval stage. The close similarity to the major malaria vector An. funestus led to investigations into the development of a molecular assay for identification of An. longipalpis. Molecular analysis of An. longipalpis from South Africa and Zambia revealed marked differences in size and nucleotide sequence in the second internal transcribed spacer (ITS2) region of ribosomal DNA between these two populations, leading to the conclusion that more than one species was being analysed. Phylogenetic analysis showed the Zambian samples aligned with An. funestus, An. vaneedeni and An. parensis, whereas the South African sample aligned with An. leesoni, a species that is considered to be more closely related to the Asian An. minimus subgroup than to the African An. funestus subgroup. Species-specific primers were designed to be used in a multiplex PCR assay to distinguish between these two cryptic species and members of the An. funestus subgroup for which there is already a multiplex PCR assay.     

Multiplex PCR and RFLP approaches for identification of rabbitfish (Siganus) species using mitochondrial gene regions

Molecular assays are described for the identification of six rabbitfish (Siganus) species. A multiplex PCR assay using primers targeting the mitochondrial cytochrome b region simultaneously identifies four species: Siganus canaliculatus, S. fuscescens, S. javus, and S. spinus. Subsequent RFLP assays of multiplex amplicons differentiate between S. virgatus and S. corallinus based on diagnostic fragments from the mitochondrial cytochrome oxidase I region. Assays were validated with known specimens demonstrating accuracy of the molecular identification. Applied to morphologically indistinguishable early developmental stages, these assays can facilitate studies on species-specific spatio-temporal patterns of larval dispersal and population connectivity to aid fishery management.