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PubMed2001

Molecular identification of Borrelia valaisiana and HGE-like Ehrlichia in Ixodes ricinus ticks sampled in north-eastern Italy: first report in Veneto region.

PCR amplification was applied to screen the presence of both Borrelia burgdorferi s.l. and Ehrlichia species in pools of field-collected Ixodes ricinus ticks. The specimens so far analysed (n = 55), grouped in 11 pools, were sampled in Feltre area (Veneto region, NE Italy). Five pools proved positive for B. valaisiana (45%) and one of them (9%) was also positive for Ehrlichia, that was further characterised as a HGE-like Ehrlichia. This is the first report of the two bacteria in the Veneto region. The pool positive for both pathogens was used to adjust a multiplex PCR assay, which allowed the detection and identification of both parasites in a single experiment. The advantages offered by this assay, when standardised, will substantially broaden the perspectives of ecological and epidemiological investigations on animal/human Lyme disease and ehrlichiosis, greatly facilitating disease surveillance and control programs.

Animals↗

PubMed1996

Coamplification in tumors of KRAS2, type 2 inositol 1,4,5 triphosphate receptor gene, and a novel human gene, KRAG.

Analysis of a region of DNA, coamplified in tumors with KRAS2, resulted in the identification of the human homologue of the mouse KRAG gene. The gene was widely expressed in a range of cell lines, tumors, and normal tissue and demonstrated a high degree of alternate splicing. A human KRAG cDNA sequence, with a structure similar to that encoded by the amplified gene in mouse Y1 adrenal carcinoma cells, was isolated by RT-PCR. The predicted amino acid similarity between the two sequences was 91%, and hydrophobicity plots suggested a structure closely resembling that of transmembrane 4 superfamily members. Identification of a PCR-based restriction fragment length polymorphism confirmed biallelic expression of KRAG but suggested allele-specific splicing differences in tumors. Northern analysis of mRNA derived from a range of tissues suggested high level expression in muscle and confirmed alternate splicing. To facilitate the analysis of exon junctions, a YAC clone encoding the genomic sequence was identified. This allowed the localization of KRAG to human chromosome 12p11.2. Isolation of one end of this nonchimeric clone demonstrated a perfect match with a 247-bp sequence within the 3' untranslated region of the type 2 1,4, 5-inositol triphosphate receptor gene. Multiplex PCR confirmed the inclusion of both genes in the KRAS2 amplicon in human malignancy, suggesting that either may contribute to the malignant phenotype.

Alleles↗

PubMed2006

Phenotypic and genotypic characteristics of methicillin/oxacillin-resistant Staphylococcus intermedius isolated from clinical specimens during routine veterinary microbiological examinations.

Methicillin/oxacillin resistance of 10 S. intermedius strains was investigated by conventional and molecular methods. The strains tested had been isolated in Germany during routine veterinary microbiological examinations of specimens from a small animal clinic between May and September 2005. Epidemiological relationships of the strains were studied by macrorestriction analysis of their chromosomal DNA using pulsed field gel electrophoresis (PFGE). Species identity of the 10 S. intermedius strains was confirmed by conventional methods and by PCR mediated amplification of S. intermedius specific segments of thermonuclease encoding gene nuc. As controls, four methicillin/oxacillin resistant S. intermedius (MRSI) strains obtained from specimens sent by four veterinarians and three selected methicillin/oxacillin sensitive S. intermedius (MSSI), also obtained from the small animal clinic, were tested. The 10 strains, representing approximately 6% of all S. intermedius isolated from the clinic throughout the time period mentioned above, and the four MRSI obtained from veterinarians, were methicillin/oxacillin and penicillin resistant using disk diffusion tests and could be cultivated on oxacillin resistant screening agar base (ORSAB). Both resistances could be confirmed by multiplex PCR detecting the resistance genes mecA and blaZ. The three MSSI were methicillin/oxacillin sensitive in all tests. Epidemiological investigation by macrorestriction analysis of the chromosomal DNA of the strains by pulsed field gel electrophoresis revealed that all 10 MRSI strains obtained from the clinic and the four MRSI strains obtained from veterinarians, in contrast to the three MSSI strains, represent identical or closely related bacterial clones possibly indicating a cross-infection of the animals in the clinic and the distribution of a single MRSI clone in the pet population.

Animals↗

PubMed2002

I(h) channels contribute to the different functional properties of identified dopaminergic subpopulations in the midbrain.

Dopaminergic (DA) midbrain neurons in the substantia nigra (SN) and ventral tegmental area (VTA) are involved in various brain functions such as voluntary movement and reward and are targets in disorders such as Parkinson's disease and schizophrenia. To study the functional properties of identified DA neurons in mouse midbrain slices, we combined patch-clamp recordings with either neurobiotin cell-filling and triple labeling confocal immunohistochemistry, or single-cell RT-PCR. We discriminated four DA subpopulations based on anatomical and neurochemical differences: two calbindin D28-k (CB)-expressing DA populations in the substantia nigra (SN/CB+) or ventral tegmental area (VTA/CB+), and respectively, two calbindin D28-k negative DA populations (SN/CB-, VTA/CB-). VTA/CB+ DA neurons displayed significantly faster pacemaker frequencies with smaller afterhyperpolarizations compared with other DA neurons. In contrast, all four DA populations possessed significant differences in I(h) channel densities and I(h) channel-mediated functional properties like sag amplitudes and rebound delays in the following order: SN/CB- --> VTA/CB- --> SN/CB+ --> VTA/CB+. Single-cell RT-multiplex PCR experiments demonstrated that differential calbindin but not calretinin expression is associated with differential I(h) channel densities. Only in SN/CB- DA neurons, however, I(h) channels were actively involved in pacemaker frequency control. In conclusion, diversity within the DA system is not restricted to distinct axonal projections and differences in synaptic connectivity, but also involves differences in postsynaptic conductances between neurochemically and topographically distinct DA neurons.

Action Potentials↗

PubMed2006

Clinical applications of molecular biology for infectious diseases.

Molecular biological methods for the detection and characterisation of microorganisms have revolutionised diagnostic microbiology and are now part of routine specimen processing. Polymerase chain reaction (PCR) techniques have led the way into this new era by allowing rapid detection of microorganisms that were previously difficult or impossible to detect by traditional microbiological methods. In addition to detection of fastidious microorganisms, more rapid detection by molecular methods is now possible for pathogens of public health importance. Molecular methods have now progressed beyond identification to detect antimicrobial resistance genes and provide public health information such as strain characterisation by genotyping. Treatment of certain microorganisms has been improved by viral resistance detection and viral load testing for the monitoring of responses to antiviral therapies. With the advent of multiplex PCR, real-time PCR and improvements in efficiency through automation, the costs of molecular methods are decreasing such that the role of molecular methods will further increase. This review will focus on the clinical utility of molecular methods performed in the clinical microbiology laboratory, illustrated with the many examples of how they have changed laboratory diagnosis and therefore the management of infectious diseases.

Journal Article↗

PubMed1995

Oligoclonality in the human CD8+ T cell repertoire in normal subjects and monozygotic twins: implications for studies of infectious and autoimmune diseases.

BACKGROUND: We have previously demonstrated CD8+ T cell clonal dominance using a PCR assay for the CDR3 length of T cell receptors belonging to a limited number of TCRBV segments/families. In this study, we have modified this approach in order to analyze more comprehensively the frequency of oligoclonality in the CD8+ T cell subset in 25 known TCRBV segments/families. In order to assess the relative roles of genes and environment in the shaping of a clonally restricted CD8+ T cell repertoire, we have analyzed clonal dominance in the CD8+ T cell population of monozygotic twins, related siblings, and adoptees. MATERIALS AND METHODS: Oligoclonality was assessed in the CD8+ T cell subsets using a multiplex PCR approach to assay for CDR3 length variation across 25 different TCRBV segments/families. Specific criteria for oligoclonality were established, and confirmed by direct sequence analysis of the PCR products. This assay was used to investigate the CD8+ T cell repertoire of 56 normal subjects, as well as six sets of monozygotic (MZ) twins. RESULTS: Seventy-two percent of normal subjects (n = 56) had evidence of oligoclonality in the CD8+ T cell subset, using well-defined criteria. Although MZ twins frequently displayed CD8+ T cell clonal dominance, the overall pattern of oligoclonality was very diverse within each twin pair. However, we occasionally observed dominant CD8+ T cell clones that were highly similar in sequence in both members of some twin pairs. Not a single example of such similarity was observed in normal controls or siblings. CONCLUSIONS: Oligoclonality of circulating CD8+ T cells is a characteristic feature of the human immune system; both host genetic factors and environment shape the pattern of oligoclonality in this T cell subset. The high frequency of this phenomenon in normal subjects provides a background with which to evaluate CD8+ T cell oligoclonality in the setting of infection or autoimmune disease. Further phenotypic and functional characterization of these clonally expanded T cells should provide insight into normal immune homeostasis.

Adolescent↗

PubMed1998

Highly accurate analysis of heterozygous loci bysingle cell PCR.

Single cell PCR is a powerful method for determining the genetic properties of individual cells. In the instance of heterozygous loci, however, preferential amplification of one allele can lead to allele drop out (ADO) of the other allele. Fortunately ADO does not occur in all amplifications, and is usually random when it does occur, with both alleles being equally susceptible to drop out. Therefore pooling of results from multiple independently amplified cells should greatly improve the analysis of diallelic loci, and the misdiagnosis rate of diallelic loci should decrease exponentially with the number of cells analysed. We have shown that this is true and that multiplex PCR allows for the simultaneous identification of a cell in a mixture of cells using microsatellite loci known to be informative, and accurate genotyping at other loci. This approach has practical applications to non-invasive prenatal diagnosis where small numbers of fetal cells in the presence of maternal cells must be both identified and genotyped at loci involved in genetic disease.

Alleles↗

PubMed2002

Subtyping of Shiga toxin 2 variants in human-derived Shiga toxin-producing Escherichia coli strains isolated in Japan.

Shiga toxin 2 (Stx2) variants have been found to exhibit not only antigenic divergence, but also differences in toxicity for tissue culture cells and animals. To clarify whether all or just a subset of Stx2 variants are important for the virulence of Shiga toxin-producing Escherichia coli, we designed PCR primers to detect and type all reported variants. We classified them into four groups according to the nucleotide sequences of the Stx2 family; for example, group 1 (G1) contains VT2vha and group 2 (G2) contains VT2d-Ount. The 120 strains of Shiga toxin-producing E. coli used in this study were isolated from humans in Japan between 1986 and 1999. Among the four variant groups, the G1 gene only was detected in 23 of the 120 clinical strains (19.2%) and all belonged to the O157 serotype. G1 is considered the most important Stx2 variant group in terms of human pathogenicity. A multiplex PCR that can detect the stx1, stx2, and G1 genes was developed as a means of rapid and easy typing to better understand the roles of the different types of Stx.

Bacterial Typing Techniques↗

PubMed2003

Analysis of polymorphism of three human Y-chromosome STR loci: DYS390, DYS392 and DYS393 in the population of northern Poland.

The study aimed at development of a multiplex PCR system for amplification of three Y-chromosome STR loci: DYS390, DYS392 and DYS393, and its application in haplotype polymorphism analysis in the population of northern Poland. Due to interactions between originally published primers, a new DYS392 primer pair was proposed. In a population of 158 unrelated males, 28 different haplotypes could be observed, 12 of which were seen only once. The haplotype diversity is 0.805. Distribution of haplotypes of the studied loci is specific to the population of northern Poland and distinguishes it from compared West-European populations. To our knowledge, this is the first report on a Y-STR multiplex system that can be analysed on native polyacrylamide gels.

Analysis of Variance↗

PubMed2002

Genomic deletions of MSH2 and MLH1 in colorectal cancer families detected by a novel mutation detection approach.

Hereditary non-polyposis colorectal cancer is an autosomal dominant condition due to germline mutations in DNA-mismatch-repair genes, in particular MLH1, MSH2 and MSH6. Here we describe the application of a novel technique for the detection of genomic deletions in MLH1 and MSH2. This method, called multiplex ligation-dependent probe amplification, is a quantitative multiplex PCR approach to determine the relative copy number of each MLH1 and MSH2 exon. Mutation screening of genes was performed in 126 colorectal cancer families selected on the basis of clinical criteria and in addition, for a subset of families, the presence of microsatellite instability (MSI-high) in tumours. Thirty-eight germline mutations were detected in 37 (29.4%) of these kindreds, 31 of which have a predicted pathogenic effect. Among families with MSI-high tumours 65.7% harboured germline gene defects. Genomic deletions accounted for 54.8% of the pathogenic mutations. A complete deletion of the MLH1 gene was detected in two families. The multiplex ligation-dependent probe amplification approach is a rapid method for the detection of genomic deletions in MLH1 and MSH2. In addition, it reveals alterations that might escape detection using conventional diagnostic techniques. Multiplex ligation-dependent probe amplification might be considered as an early step in the molecular diagnosis of hereditary non-polyposis colorectal cancer.

Adaptor Proteins, Signal Transducing↗

PubMedDate not supplied

[Prevalence of bacteria in children with otitis media with effusion].

OBJECTIVES: 1) To determine the prevalence of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis in middle ear effusions of children with otitis media with effusion undergoing myringotomy; 2) to compare the results obtained by culture and PCR; and 3) to determine the susceptibility of bacterial isolates to penicillin. METHODS: We analyzed 128 middle ear effusion specimens from 75 children (age = 11 months to 10 years; mean = 34.7 months). Patients with recurrent otitis media had documented middle ear effusion for >/= 6 weeks, and chronic otitis media with effusion for >/= 3 months. The patients had no signs of acute otitis media or respiratory tract infection and were not on antibiotic therapy. Aspiration was done through tympanocentesis with an Alden-Senturia trap. Bacteriological studies were initiated less than 15 minutes after specimen collection. Part of the sample was stored at -20oC for later multiplex PCR analysis. Statistical analysis employed McNemar's (Chi2) test. RESULTS: Bacteria were cultured in 32 (25.1%) out of 128 samples and the pathogens under investigation were found in 25 (19.6%). PCR was positive for bacteria in 73 (57.0%) specimens: 50 (39.1%) for H. influenzae, 16 (12.5%) for S. pneumoniae, and 13 (10.2%) for M. catarrhalis. All the culture-positive samples were PCR-positive, but 48 (65.7%) of the PCR-positive specimens were culture-negative. PCR was significantly more sensitive than culture (p < 0.01) to identify bacteria. Resistance to penicillin was as follows: M. catarrhalis = 100%; S. pneumoniae = 62.5% and H. influenzae = 23% of the isolates. CONCLUSIONS: The prevalence of bacteria in otitis media with effusion in a group of Brazilian children was similar to that reported for other countries. H. influenzae was the most frequent microorganism observed. This suggests that bacteria may play a role in the pathogenesis of otitis media with effusion. In addition, PCR was more sensitive to detect bacteria in middle ear effusion as compared to conventional culture methods. Penicillin resistance was similar to that reported for other countries for pneumococci and moraxella, but beta-lactamase production by H. influenza was lower than that reported for other countries.

Child↗

PubMed1999

Selection of RAPD marker for growth of seedlings at low temperature in rice.

We have developed a polymerase chain reaction (PCR)-based assay that could effectively reduce the time period required to screen and select the cold tolerance gene of rice seedlings under field conditions. The two specific random amplified polymorphic DNA (RAPD) fragments for the assay were identified on the basis of quantitative trait loci (QTL) analysis which were found to be tightly linked to cold sensitivity. The two RAPD fragments, OPT8(600) in the cold sensitivity rice cultivar 'Dular (indica)' and OPU20(1200) in the resistance rice cultivar 'Toyohatamochi (japonica)', were identified after screening 11 RAPD fragments using 2 random primers on the genomic DNAs of 'Dular' and 'Toyohatamochi'. These primers, when used in a multiplexed PCR, specifically amplified a 0.6 kb and a 1.2 kb fragment in the sensitive and resistant rice cultivars, respectively. When this assay was performed on the genomic DNAs of 16 japonica, 3 Tongil (indica/ japonica), and 2 indica rice cultivars, the primers amplified a 0.6 kb fragment in all of the cold sensitivity rice cultivars or 1.2 kb fragment in all of the resistance ones. These markers can be of potential use in the marker-assisted selection (MAS) for cold tolerance in rice seedling. As screening for resistance can now be conducted independent of the availability of low temperature, the breeding of cold tolerance cultivars can be hastened.

Acclimatization↗

PubMed2002

False-positive Y-microdeletion result for a fertile male caused by an alteration under a PCR primer.

The pathogenic relationship between the presence of Y chromosomal microdeletions and male infertility is unclear. Nevertheless, a causal relationship is thought to be probable when loci are shown to be deleted in infertile males but are present in fertile males. Polymerase chain reaction (PCR) analysis of the Y chromosome is now routinely performed in the evaluation of the infertile male, although, until recently, there has been no consensus on how the diagnosis should be performed and which loci or markers should be analysed. The European Academy of Andrology (EAA) published guidelines for the molecular diagnosis of Y chromosomal microdeletions in 1999. Following these guidelines, our laboratory developed assays that incorporated the suggested primer pairs for the recommended Sequence Tagged Sites (STS). A number of fertile (n = 117), infertile (n = 17) and unknown samples (n = 20) were tested in our laboratory as part of the validation to provide a clinical assay. Two multiplex PCR assays were optimized, each of which examined STS markers in the centre of the AZFa, b and c regions of the Y chromosome. We correctly identified all but one of the 154 samples (according to the expected result based on fertility or previous testing at another laboratory). A single equivocal result was observed for a sample obtained from a known fertile male who appeared to be deleted for a single marker, sY84, in the AZFa region but not the adjacent marker, sY86. Follow-up analysis showed that proximal and distal markers within the same region (sY82 and sY98) were also present. Sequencing the region flanking and including the sY84 primer set revealed a single base alteration under the reverse primer, which probably caused the amplification failure. Furthermore, the sY84 sequence itself was present, as was the flanking sequence 50 bp on either side of both primers. This observation underlines the importance of using at least two closely linked STS markers for the reliable diagnosis of Y chromosome microdeletions as proposed by the EAA guidelines.

Base Sequence↗

PubMed1996

Molecular dissection of corticosteroid action in the rat hippocampus. Application of the differential display techniques.

Both adrenal steroids and glutamate are crucial for hippocampal cell viability. In order to identify adrenal steroid- and glutamate-responsive genes controlling hippocampal cell viability, we have used the PCR-based differential display method. We have described the characteristics of this technique and how it can be automated. Using differential display, we have identified a number of rat hippocampal genes of which the expression is affected by a combination of the glutamate analog kainic acid and adrenalectomy. Administration of kainic acid or removal of the adrenals alone gave a limited number of differentially displayed genes. Therefore, our results indicate that the main mode of corticosteroid receptor-controlled gene expression in the hippocampus is interaction with other transcription factors (e.g., CREB, AP-1) and not by binding to hormone-responsive elements of corticosterone-specific genes. Characterization by multiplex PCR experiments of a differentially displayed fragment of which the expression is increased by the combination of kainic acid and adrenalectomy confirmed our differential display results. Further characterization by DNA sequence analysis of the corresponding full-length cDNA clone revealed a gene product with 91.4% sequence identity with the mouse transcription factor KROX-20, suggesting that we have cloned the rat homolog. This finding suggests a role of KROX-20 in corticosteroid- and kainic acid-controlled hippocampal plasticity.

Adrenalectomy↗

PubMed1999

Frequent allelic loss on chromosome 9 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common malignancy worldwide and highly associated with chronic virus-B or -C infection and cirrhosis. Molecular studies have shown high frequency of loss of heterozygosity (LOH) in some specific chromosome regions, but LOH on chromosome 9 in HCC has not been thoroughly investigated. In our investigation of chromosome 9 with 19 polymerase-chain-reaction (PCR)-based polymorphic microsatellite markers, 30 of 48 HCC tissue samples (63%) had LOH, and a distinct common deletion region and a region of loss were identified. The first region was located at the 9p21 region and the minimal deletion region was located between loci D9S1747 and D9S1748. This is a region of approximately 200 kb which includes the p16 tumor-suppressor gene. A region of loss was located on 9p13 to 9q33. The putative tumor-suppressor gene for nevoid-basal-cell-carcinoma syndrome (NBCCS) at 9q22.3 resides within this region. In addition to LOH, 4 HCC cases showed possible homozygous deletions at 9p21 with markers D9S1748, D9S1752 and D9S171 by multiplex PCR analysis. In 3 cases, the minimal region of possible homozygous deletion was approximately 300 kb and was defined between markers D9S1747 and D9S1752. Since this deletion region includes both the p15 and the p16 tumor-suppressor genes, these genes were possibly inactivated by homozygous deletion in HCC. In addition, a second region of possible homozygous deletion was present on the centromeric side of 9p21. However, these changes are not associated with age, gender, size or tumor-cell differentiation. Our data also suggest that inactivation of the p16 and the p15 genes and the possibility of other unknown tumor-suppressor genes located on these defined deleted regions of chromosome 9 may be involved in the pathogenesis of HCC.

Adolescent↗

PubMed1999

GABA(A) receptor-mediated miniature postsynaptic currents and alpha-subunit expression in developing cortical neurons.

Previous studies have described maturational changes in GABAergic inhibitory synaptic transmission in the rodent somatosensory cortex during the early postnatal period. To determine whether alterations in the functional properties of synaptically localized GABA(A) receptors (GABA(A)Rs) contribute to development of inhibitory transmission, we used the whole cell recording technique to examine GABAergic miniature postsynaptic currents (mPSCs) in developing cortical neurons. Neurons harvested from somatosensory cortices of newborn mice showed a progressive, eightfold increase in GABAergic mPSC frequency during the first 4 wk of development in dissociated cell culture. A twofold decrease in the decay time of the GABAergic mPSCs, between 1 and 4 wk, demonstrates a functional change in the properties of GABA(A)Rs mediating synaptic transmission in cortical neurons during development in culture. A similar maturational profile observed in GABAergic mPSC frequency and decay time in cortical neurons developing in vivo (assessed in slices), suggests that these changes in synaptically localized GABA(A)Rs contribute to development of inhibition in the rodent neocortex. Pharmacological and reverse transcription-polymerase chain reaction (RT-PCR) studies were conducted to determine whether changes in subunit expression might contribute to the observed developmental alterations in synaptic GABA(A)Rs. Zolpidem (300 nM), a subunit-selective benzodiazepine agonist with high affinity for alpha1-subunits, caused a reversible slowing of the mPSC decay kinetics in cultured cortical neurons. Development was characterized by an increase in the potency of zolpidem in modulating the mPSC decay, suggesting a maturational increase in percentage of functionally active GABA(A)Rs containing alpha1 subunits. The relative expression of alpha1 versus alpha5 GABA(A)R subunit mRNA in cortical tissue, both in vivo and in vitro, also increased during this same period. Furthermore, single-cell RT-multiplex PCR analysis revealed more rapidly decaying mPSCs in individual neurons in which alpha1 versus alpha5 mRNA was amplified. Together these data suggest that changes in alpha-subunit composition of GABA(A)Rs contribute to the maturation of GABAergic mPSCs mediating inhibition in developing cortical neurons.

Animals↗

PubMed2000

Differential diagnosis of Taenia saginata and Taenia solium infection by PCR.

We have designed species-specific oligonucleotides which permit the differential detection of two species of cestodes, Taenia saginata and Taenia solium. The oligonucleotides contain sequences established for two previously reported, noncoding DNA fragments cloned from a genomic library of T. saginata. The first, which is T. saginata specific (fragment HDP1), is a repetitive sequence with a 53-bp monomeric unit repeated 24 times in direct tandem along the 1, 272-bp fragment. From this sequence the two oligonucleotides that were selected (oligonucleotides PTs4F1 and PTs4R1) specifically amplified genomic DNA (gDNA) from T. saginata but not T. solium or other related cestodes and had a sensitivity down to 10 pg of T. saginata gDNA. The second DNA fragment (fragment HDP2; 3,954 bp) hybridized to both T. saginata and T. solium DNAs and was not a repetitive sequence. Three oligonucleotides (oligonucleotides PTs7S35F1, PTs7S35F2, and PTs7S35R1) designed from the sequence of HDP2 allowed the differential amplification of gDNAs from T. saginata, T. solium, and Echinococcus granulosus in a multiplex PCR, which exhibits a sensitivity of 10 pg.

Animals↗

PubMed1997

PCR assay based on a microsatellite-containing locus for detection and quantification of Epichloë endophytes in grass tissue.

A PCR assay which allows detection and quantification of Epichloë endophytes in tissues of the grass Bromus erectus is described. PCR with specific primers flanking a microsatellite-containing locus (MS primers) amplified fragments 300 to 400 bp in length from as little as 1.0 pg of fungal genomic DNA in 100 ng of DNA from infected plant material. When annealing temperatures were optimized, all Epichloë and Acremonium strains tested, representing many of the known taxonomic groups, yielded an amplification product, indicating that the MS primers may be useful for in planta detection of a variety of related species, including agronomically important Acremonium coenophialum and Acremonium lolii. No fragments were generated from DNA isolates from uninfected plant material or from unrelated fungi isolated from B. erectus. For diagnostic applications, a B. erectus-specific primer pair was designed for use in multiplex PCR to allow simultaneous amplification of plant and fungal DNA sequences, providing an internal control for PCR failure caused by inhibitory plant compounds present in DNA extracts. For quantitative applications, a heterologous control template in primer binding sites complementary to the MS primers was constructed for use in competitive PCR, allowing direct quantification of Epichloë in plant DNA extracts. The fungal DNA present in infected leaves of B. erectus between 1 and 20 pg per 100 ng of leaf DNA, but the amounts of fungal DNA present in the sheath and blade of a given leaf were correlated, indicating that the degree of infection varied between plant individuals but that leaves were colonized in a uniform way.

Acremonium↗