Nested PCR Assays for Detection of Monilinia fructicola in Stone Fruit Orchards and Botryosphaeria dothidea from Pistachios in California

Nested polymerase chain reaction (PCR) assays were developed based on microsatellite regions for detection of Monilinia fructicola, the causal agent of brown rot of stone fruits, and Botryosphaeria dothidea, the causal agent of panicle and shoot blight of pistachio. The nested PCR primers specific to M. fructicola were developed based upon the sequence of a species‐specific DNA fragment amplified by microsatellite primer M13. The external and internal primer pairs EMfF + EMfR and IMfF + IMfR amplified a 571‐ and a 468‐bp fragment, respectively, from M. fructicola, but not from any other fungal species present in stone fruit orchards. The nested PCR primer pairs specific to B. dothidea were developed based upon the sequence of a species‐specific 1330‐bp DNA fragment amplified by microsatellite primer T3B. The external and internal primer pairs EBdF + EBdR and IBdF + IBdR amplified a 701‐ and a 627‐bp fragment, respectively, from B. dothidea, but not from any other fungal species associated with pistachio. The nested PCR assays were sensitive enough to detect the specific fragments in 1 fg of M. fructicola or B. dothidea DNA or in the DNA from only two conidia of M. fructicola or B. dothidea. The nested PCR assays could detect small numbers of M. fructicola conidia caught on spore‐trap tapes and detect visible infections of B. dothidea in pistachio tissues. Microsatellite regions with high numbers of copies are widely dispersed in eukaryotic genomes. The results of this study indicate that microsatellite regions could be useful in developing highly sensitive PCR detection systems for phytopathogenic fungi.     

Assessment and modification of degenerate qPCR primers that amplify functional genes from etheneotrophs and vinyl chloride-assimilators

Aims: Degenerate qPCR primer sets that target the functional genes etnC and etnE in etheneotrophs and vinyl chloride‐assimilating bacteria were assessed and modified in an effort to improve performance. Methods and Results: Functional gene abundance in four pure cultures was estimated by qPCR using novel (MRTC and MRTE) and existing (RTC and RTE) degenerate primer sets and compared to abundances estimated with nondegenerate gene‐specific primers (GSPs). Functional gene abundance in groundwater DNA extracted from several contaminated sites was also estimated with MRTC and MRTE primers. Conclusions: MRTC primers displayed significantly improved etnC quantification in both pure cultures and environmental samples. Significance and Impact of the Study: Application of MRTC and MRTE primer sets will enhance microbial ecology studies involving etheneotrophs and qPCR analyses that support vinyl chloride bioremediation strategies.     

Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies

16S ribosomal RNA gene (rDNA) amplicon analysis remains the standard approach for the cultivation-independent investigation of microbial diversity. The accuracy of these analyses depends strongly on the choice of primers. The overall coverage and phylum spectrum of 175 primers and 512 primer pairs were evaluated in silico with respect to the SILVA 16S/18S rDNA non-redundant reference dataset (SSURef 108 NR). Based on this evaluation a selection of ‘best available’ primer pairs for Bacteria and Archaea for three amplicon size classes (100–400, 400–1000, ≥1000 bp) is provided. The most promising bacterial primer pair (S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21), with an amplicon size of 464 bp, was experimentally evaluated by comparing the taxonomic distribution of the 16S rDNA amplicons with 16S rDNA fragments from directly sequenced metagenomes. The results of this study may be used as a guideline for selecting primer pairs with the best overall coverage and phylum spectrum for specific applications, therefore reducing the bias in PCR-based microbial diversity studies.     

Universal primers for fluorescent labelling of PCR fragments--an efficient and cost-effective approach to genotyping by fluorescence

Directly labelling locus‐specific primers for microsatellite analysis is expensive and a common limitation to small‐budget molecular ecology projects. More cost‐effective end‐labelling of PCR products can be achieved through a three primer PCR approach, involving a fluorescently labelled universal primer in combination with modified locus‐specific primers with 5′ universal primer sequence tails. This technique has been widely used but has been limited largely due to a lack of available universal primers suitable for co‐amplifying large numbers of size overlapping loci and without requiring locus‐specific PCR conditions to be modified. In this study, we report a suite of four high‐performance universal primers that can be employed in a three primer PCR approach for efficient and cost‐effective fluorescent end‐labelling of PCR fragments. Amplification efficiency is maximized owing to high universal primer Tm values (approximately 60+ °C) that enhance primer versatility and enable higher annealing temperatures to be employed compared with commonly used universal primers such as M13. We demonstrate that these universal primers can be combined with multiple fluorophores to co‐amplify multiple loci efficiently via multiplex PCR. This method provides a level of multiplexing and PCR efficiency similar to microsatellite fluorescent detection assays using directly labelled primers while dramatically reducing project costs. Primer performance is tested using several alternative PCR strategies that involve both single and multiple fluorophores in single and multiplex PCR across a wide range of taxa.     

Molecular Characterization of Aster Yellows Phytoplasma Associated with Valerian and Sowthistle Plants by PCR–RFLP Analyses

In Alberta, Canada, valerian grown for medicinal purposes and sowthistle, a common weed, showed typical aster yellows symptoms. Molecular diagnosis was made using a universal primer pair (P1 / P7) designed to amplify the entire 16S rRNA gene and the 16 / 23S intergenic spacer region in a direct polymerase chain reaction (PCR) assay. This primer pair amplified the DNA samples from valerian and sowthistle and reference controls (AY‐27, CP, PWB, AY of canola, LWB). They produced the expected PCR products of 1.8 kb, which were diluted and used as templates in a nested PCR. Two primer pairs R16F2n / R2 and P3 / P7 amplified the DNA templates giving PCR products of 1.2 and 0.32 kb, respectively. No PCR product was obtained with either set of primers and DNA isolated from healthy plants. Restriction fragment length polymorphism (RFLP) was used to analyse the partial 16S rDNA sequences (1.2 kb) of all phytoplasma DNA samples after restriction with four endonucleases (AluI, HhaI, MseI and RsaI). The restriction patterns of these strains were found to be identical with the RFLP pattern of the AY phytoplasma reference control (AY‐27 strain). Based on the RFLP data, the two strains are members of subgroup A of the AY 16Sr1 group. We report here the first molecular study on the association of AY phytoplasmas with valerian and sowthistle plants.     

Don't put all your eggs in one basket: a cost‐effective and powerful method to optimize primer choice for rRNA environmental community analyses using the Fluidigm Access Array

With the increasing democratization of high‐throughput sequencing (HTS) technologies, along with the concomitant increase in sequence yield per dollar, many researchers are exploring HTS for microbial community ecology. Many elements of experimental design can drastically affect the final observed community structure, notably the choice of primers for amplification prior to sequencing. Some targeted microbes can fail to amplify due to primer‐targeted sequence divergence and be omitted from obtained sequences, leading to differences among primer pairs in the sequenced organisms even when targeting the same community. This potential source of taxonomic bias in HTS makes it prudent to investigate how primer choice will affect the sequenced community prior to investing in a costly community‐wide sequencing effort. Here, we use Fluidigm's microfluidic Access Arrays (IFC) followed by Illumina^® MiSeq Nano sequencing on a culture‐derived local mock community to demonstrate how this approach allows for a low‐cost combinatorial investigation of primer pairs and experimental samples (up to 48 primer pairs and 48 samples) to determine the most effective primers that maximize obtained communities whilst minimizing taxonomic biases.     

The effect of nutrient deposition on bacterial communities in Arctic tundra soil

The microbial communities of high‐latitude ecosystems are expected to experience rapid changes over the next century due to climate warming and increased deposition of reactive nitrogen, changes that will likely affect microbial community structure and function. In moist acidic tundra (MAT) soils on the North Slope of the Brooks Range, Alaska, substantial losses of C and N were previously observed after long‐term nutrient additions. To analyse the role of microbial communities in these losses, we utilized 16S rRNA gene tag pyrosequencing coupled with community‐level physiological profiling to describe changes in MAT bacterial communities after short‐ and long‐term nutrient fertilization in four sets of paired control and fertilized MAT soil samples. Bacterial diversity was lower in long‐term fertilized plots. The Acidobacteria were one of the most abundant phyla in all soils and distinct differences were noted in the distributions of Acidobacteria subgroups between mineral and organic soil layers that were also affected by fertilization. In addition, Alpha‐ and Gammaproteobacteria were more abundant in long‐term fertilized samples compared with control soils. The dramatic increase in sequences within the Gammaproteobacteria identified as Dyella spp. (order Xanthomonadales) in the long‐term fertilized samples was confirmed by quantitative PCR (qPCR) in several samples. Long‐term fertilization was also correlated with shifts in the utilization of specific substrates by microbes present in the soils. The combined data indicate that long‐term fertilization resulted in a significant change in microbial community structure and function linked to changes in carbon and nitrogen availability and shifts in above‐ground plant communities.     

Changes of 5＇ Terminal Nucleotides of PCR Primers Causing Variable T-A Cloning Efficiency

T-A cloning takes advantage of the unpaired adenosyl residue added to the 3＇ terminus of amplified DNAs by Taq and other thermostable DNA polymerase and uses a Ilnearlzed plasmld vector with a protruding 3＇ thymldylate residue at each of Its 3＇ termini to clone polymerase chain reaction （PCR）-derived DNA fragments. It Is a simple, reliable, and efficient Ilgatlon-dependent cloning method for PCR products, but the drawback of variable cloning efficiency occurs during application. In the present work, the relationship between variable T-A cloning efficiency and the different 5＇ end nucleotlde base of primers used In PCR amplification was studied. The results showed that different cloning efficiency was obtained with different primer pairs containing A, T, C and G at the 5＇ terminus respectively. The data shows that when the 5＇ end base of primer pair was adenosyl, more white colonies could be obtained In cloning the corresponding PCR product In comparison with other bases. And the least white colonies were formed when using the primer pair with 5＇ cytldylate end. The gluanylate end primers resulted In almost the same cloning efficiency In the white colonies amount as the thymldylate end primer did, and this efficiency was much lower than that of adenosyl end primers. This presumably is a consequence of variability In 3＇dA addition to PCR products mediated by Taq polymerase. Our results offer instructions for primer design for researchers who choose T-A cloning to clone PCR products.     

A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.)

A multiplex nested PCR assay was developed by optimizing reaction components and reaction cycling parameters for simultaneous detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma (Group 16Sr V‐C) causing little leaf and bunchy top in white jute (Corchorus capsularis). Three sets of specific primers viz. a CoGMV specific (DNA‐A region) primer, a 16S rDNA universal primer pair P1/P7 and nested primer pair R16F2n/R2 for phytoplasmas were used. The concentrations of the PCR components such as primers, MgCl₂, Taq DNA polymerase, dNTPs and PCR conditions including annealing temperature and amplification cycles were examined and optimized. Expected fragments of 1 kb (CoGMV), 674 bp (phytoplasma) and 370 bp (nested R16F2n/R2) were successfully amplified by this multiplex nested PCR system ensuring simultaneous, sensitive and specific detection of the phytoplasma and the virus. The multiplex nested PCR provides a sensitive, rapid and low‐cost method for simultaneous detection of jute little leaf phytoplasma and CoGMV. Based on BLASTn analyses, the phytoplasma was found to belong to the Group 16Sr V‐C.

Significance and Impact of the Study

Incidence of phytoplasma diseases is increasing worldwide and particularly in the tropical and subtropical world. Co‐infection of phytoplasma and virus(s) is also common. Therefore, use of single primer PCR in detecting these pathogens would require more time and effort, whereas multiplex PCR involving several pairs of primers saves time and reduces cost. In this study, we have developed a multiplex nested PCR assay that provides more sensitive and specific detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma in white jute simultaneously. It is the first report of simultaneous detection of CoGMV and a phytoplasma in Corchorus capsularis by multiplex nested PCR.     

Development of Streptococcus gordonii‐specific quantitative real‐time polymerase chain reaction primers based on the nucleotide sequence of rpoB

In this study, Streptococcus gordonii‐specific quantitative real‐time polymerase chain reaction (qPCR) primers, RTSgo‐F2/RTSgo‐R2, were developed based on the nucleotide sequences of RNA polymerase β‐subunit gene (rpoB). The specificity of the RTSgo‐F2/RTSgo‐R2 primers was assessed by conventional PCR on 99 strains comprising 63 oral bacterial species, including the type strain and eight clinical isolates of S. gordonii. PCR products were amplified from the genomic DNAs of only S. gordonii strains. The qPCR primers were able to detect as little as 40 fg of S. gordonii genomic DNA at a cycle threshold value of 33. These findings suggest that these qPCR primers detect S. gordonii with high specificity and sensitivity.     

Isolation and characterization of polymorphic microsatellite markers from European pear (Pyrus communis L.)

This study reports the development and characterization of 19 microsatellite primer pairs developed from genomic DNA of European pear (Pyrus communis) and their transferability to other Pyrus and Malus material. The primers were designed from two different genomic libraries enriched for di‐ and trinucleotide repeats. When tested in six P. communis cultivars and 15 other Pyrus species, 13 primers revealed single‐locus polymorphism and six showed more complex patterns that suggest multiple loci. Two to 18 alleles were detected per locus and two primer pairs were sufficient to discriminate all accessions. Transferability of nine primer pairs to Malus was demonstrated through amplification of discrete products in two accessions.     

Development of novel polymerase chain reaction (PCR) based microsatellite markers in Armillaria gallica by cross‐species amplification and species‐specific cloning

Cross‐species PCR amplification of Armillaria mellea group taxa with previously reported A. ostoyae microsatellite markers, indicative of flanking sequence conservation, was exploited for the species‐specific isolation of simple sequence repeat (SSR) motifs from A. gallica. Six SSR motifs were sequence characterized from cloned PCR fragments generated with primers previously developed from A. ostoyae. Five novel primer pairs, designed from motif flanking regions, allowed for improved, efficient amplification in this species. One original A. ostoyae primer pair was used directly. Polymorphims were observed at wide geographical levels only. Relative cross‐species amplification intensities generally supported the currently accepted molecular phylogeny of this group.     

‘Candidatus Phytoplasma asteris’ and ‘Candidatus Phytoplasma aurantifolia’, New Phytoplasma Species Infecting Apple Trees in Iran

During several surveys in extensive areas in central Iran, apple trees showing phytoplasma diseases symptoms were observed. PCR tests using phytoplasma universal primer pairs P1A/P7A followed by R16F2n/R16R2 confirmed the association of phytoplasmas with symptomatic apple trees. Nested PCR using 16SrX group‐specific primer pair R16(X)F1/R1 and aster yellows group‐specific primer pairs rp(I)F1A/rp(I)R1A and fTufAy/rTufAy indicated that apple phytoplasmas in these regions did not belong to the apple proliferation group, whereas aster yellows group‐related phytoplasmas caused disease on some trees. Restriction fragment length polymorphism (RFLP) analyses using four restriction enzymes (HhaI, HpaII, HaeIII and RsaI) and sequence analyses of partial 16S rRNA and rp genes demonstrated that apple phytoplasma isolates in the centre of Iran are related to ‘Ca. Phytoplasma asteris’ and ‘Ca. Phytoplasma aurantifolia’. This is the first report of apples infected with ‘Ca. Phytoplasma asteris’ in Iran and the first record from association of ‘Ca. Phytoplasma aurantifolia’ with apples worldwide.     

Improved efficiency and robustness in qPCR and multiplex end-point PCR by twisted intercalating nucleic acid modified primers

We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 5'-end. In qPCR, the 5'-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5'-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5'-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5'-o-TINA modified primers allows for a further reduction (>45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5'-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5'-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions.     

A Plating‐PCR Technique for Detection and Quantification of the Biological Control Agent Agrobacterium vitis Strain E26 in Soil under Controlled Conditions

Agrobacterium vitis strain E26 is a promising biocontrol agent of grapevine crown gall, an economically important disease of grape worldwide. In this report, we developed a Plating‐PCR method that allows specific detection and quantification of E26 by combining classical microbiological techniques with molecular tools. Random amplified polymorphic DNA fingerprints were used to differentiate E26 from other A. vitis strains. A differentially amplified fragment from E26 was sequenced and characterized as a sequence characterized amplified region (SCAR) marker. Two primer pairs were then designed and evaluated for their specificity against E26. One of the two SCAR primer pairs, 740F/R, was further selected for specific detection of strain E26. A plating assay coupled to PCR with the SCAR primers 740F/R allowed the assessment of population dynamics of E26 in non‐sterile grape rhizosphere soil under controlled conditions.     

Molecular identification of the economically important freshwater mussels (Mollusca–Bivalvia–Unionoida) of Thailand: developing species‐specific markers from AFLPs

Shells of certain freshwater mussel (Unionoida) species are highly demanded and serve as raw material for a range of decorative and pharmaceutical products. In Thailand, most animals for this purpose are currently harvested from wild populations, with unionoid culture still being in its infancy. Whilst reliable species identification is a prerequisite for developing a large‐scale industry, identification by morphological means is hampered by extensive phenotypic plasticity and poor knowledge of species delimitations. To facilitate alternative molecular identification, we developed species‐specific markers for the three Thai unionoids with considerable economic potential (CEP): that is, Chamberlainia hainesiana, Hyriopsis desowitzi and Hyriopsis myersiana. For this purpose, amplified fragment length polymorphism (AFLP) fingerprints using 24 specific primer pairs were generated for eight samples of each CEP species and four samples of the closely related, non‐CEP species Contradens contradens. Cloning and sequencing of 13 CEP species‐specific AFLP bands revealed fragment collision at three occasions. In total, 16 species‐specific primer pairs were designed and tested on 92 Thai specimens spanning seven species and four genera. Thereby, specificity of (1) three primers to C. hainesiana, (2) one primer to H. desowitzi + Hyriopsis bialata, (3) one primer to H. myersiana + H. bialata and (4) four primers to all three Hyriopsis species tested was confirmed. Respective multiplex PCR protocols are provided. The developed primers enable cheap, quick and reliable identification of the Thai CEP species by one to three PCRs and offer a tool for a range of additional applications within mussel culture and ecological and evolutionary research on these important organisms.     

Species‐specific detection of Candida tropicalis using evolutionary conserved intein DNA sequences

Inteins (internal proteins) are self‐splicing transportable genetic elements present in conserved regions of housekeeping genes. The study highlights the importance of intein as a potential diagnostic marker for species‐specific identification of Candida tropicalis, a rapidly emerging opportunistic human pathogen. Initial steps of primer validation, sequence alignment, phylogenetic tree analysis, gel electrophoresis and real‐time polymerase chain reaction (PCR) assays were performed to confirm the specificity of the designed primers. The primers were selective for C. tropicalis with 100% inclusivity and showed no cross‐species or cross‐genera matches. The established technique is a prototype for developing multifaceted PCR assays and for point‐of‐care testing in near future.

Significance and Impact of the Study

Development of molecular markers for specific detection of microbial pathogens using real‐time polymerase chain reaction (PCR) is an appealing and challenging technique. A real‐time PCR is an emerging technology frequently used to detect the aetiologic agents. In recent times, designing species‐specific primers for pathogen detection is gaining momentum. The method offers rapid, accurate and cost‐effective strategy to identify the target, thus providing sufficient time to instigate appropriate chemotherapy. The study highlights the use of intein DNA sequence as molecular markers for species‐specific identification of Candida tropicalis. The study also offers a prototype model for developing multifaceted PCR assays using intein DNA sequences, and provides a developmental starting point for point‐of‐care testing in near future.     

Primer evaluation and adaption for cost-efficient SYBR Green-based qPCR and its applicability for specific quantification of methanogens

In the present study nine promising primer sets, targeting Archaea and methanogenic Archaea in particular, were evaluated in silico, in vitro and in situ concerning specificity, accuracy and applicability in end-point (ep-) and especially quantitative (q-)PCR research. The main goal was to adapt and evaluate already adapted primer sets, which were partially designed in combination with TaqMan probes, in substantially cheaper SYBR Green-based qPCR applications. An initial 16S rRNA gene bank-based in silico evaluation revealed high coverage potentials for all primers within targeted groups, ranging from 71 to 90 %, except the Methanosaeta specific set showing a low potential of 37 %. Mentionable cross-reacting potentials could be detected for the Methanothermobacter, Methanomicrobiales and Methanoculleus sets. The in vitro evaluation with selected reference organisms revealed a specific behavior for most primer sets, while the Methanosarcina and Methanothermobacter sets showed most problematic cross-reactions in epPCR application. We were able to show that primers for detecting the total archaeal community, methanogenic orders Methanosarcinales, Methanobacteriales, Methanococcales and the genus Methanoculleus performed in a highly specific way and allowed an accurate quantification of targeted organisms without the use of expensive TaqMan probes. However, primer pairs designed for detecting Methanomicrobiales, Methanothermobacter, Methanosarcina and Methanosaeta are not suitable for SYBR Green applications. The reliability of in situ quantifications was assessed for a typical methanogenic community, derived from a thermophilic fermenter, and confirmed via denaturing gradient gel band quantification and sequencing. Thereby, we revealed high abundances of methanogenic Archaea, mainly comprising Methanoculleus and Methanosarcinales, while Methanobacteriales only formed a minor fraction.     

PRaTo: a web-tool to select optimal primer pairs for qPCR

An essential pre-requisite to perform sound quantitative real-time polymerase chain reaction (qPCR) assays is to design outstanding primer pairs. This means they must have a good efficiency and be not prone to produce multiple amplicons or primer dimer products. To circumvent these issues, several softwares are available to help primer design. Although satisfactory computer-aided primer design tools are available for standard PCR, less efforts were done to provide specific methods for selection of optimal primer pairs for qPCR. We have developed PRaTo a web-based tool that enables checking and ranking of primers pairs for their attitude to perform optimally and reliably when used in qPCR experiments. PRaTo is available at http://prato.daapv.unipd.it.