Development of a highly sensitive nested-PCR procedure using a single closed tube for detection of Erwinia amylovora in asymptomatic plant material

A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in E. amylovora hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 microl of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of E. amylovora in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity.     

Development of a Highly Sensitive Nested-PCR Procedure Using a Single Closed Tube for Detection of Erwinia amylovora in Asymptomatic Plant Material

A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in E. amylovora hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 μl of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of E. amylovora in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity.     

Direct polymerase chain reaction detection of ropy Pediococcus damnosus strains in wine

AIMS: Glucan-producing strains of Pediococcus damnosus are considered as spoilage micro-organisms because synthesis of glucan leads to an unacceptable viscosity of wine. In this report, we present a polymerase chain reaction (PCR) procedure to detect the presence of such strains in wines. METHODS AND RESULTS: We developed a direct DNA isolation method from the wine microflora using polyvinylpyrrolidone in order to decrease the polyphenolic concentration. The sequence of the plasmid involved in glucan production allowed the design of a primer pair usable for a specific and sensitive PCR procedure, leading to the amplification of a 563-bp fragment. CONCLUSION: The detection limit in wine was 102 cfu ml-1. The detection sensitivity could be increased by using a second primer pair in nested PCR assays. SIGNIFICANCE AND IMPACT OF THE STUDY: The method proved to be efficient for the early and sensitive detection of ropy Ped. damnosus strains during wine-making. Time-consuming culture and colony isolation steps are no longer needed.     

Detection of Fusarium culmorum in wheat by a surface plasmon resonance-based DNA sensor

A surface plasmon resonance (SPR) sensor based on DNA hybridization has been developed for the detection of Fusarium culmorum, a fungal pathogen of cereals. A 0.57 kbp DNA fragment of F. culmorum was amplified by specific primers and a 25-mer oligonucleotide probe was selected within the sequence of the PCR amplicon. After biotinilation, the probe was immobilized on a streptavidin sensor chip and tested for biospecific interaction with PCR products of F. culmorum. The effect of denaturating agents (formamide and urea) and ionic strength (NaCl) on hybridization efficiency of double-stranded PCR products with the immobilized probe and the specificity of the probe were investigated. The SPR biosensor was successfully used for the detection of F. culmorum in culture material of different strains and in naturally infected wheat samples. Tested on fungal cultures, it showed a good selectivity for F. culmorum against other species of either Fusarium or other fungal genera. A background signal was observed in wheat samples strictly depending on the DNA amount of the testing matrix. Testing 30 ng of durum wheat DNA the detection limit was 0.06 pg of F. culmorum DNA. The developed PCR-SPR assay allowed to detect F. culmorum with sensitivity and specificity higher than gel-electrophoresis analysis.     

A nested-PCR assay for detection of Xylella fastidiosa in citrus plants and sharpshooter leafhoppers

AIMS: Detection of Xylella fastidiosa in citrus plants and insect vectors. METHODS AND RESULTS: Chelex 100 resin matrix was successfully standardized allowing a fast DNA extraction of X. fastidiosa. An amplicon of 500 bp was observed in samples of citrus leaf and citrus xylem extract, with and without symptoms of citrus variegated chlorosis, using PCR with a specific primer set indicating the presence of X. fastidiosa. The addition of insoluble acid-washed polyvinylpyrrolidone (PVPP) prior to DNA extraction of insect samples using Chelex 100 resin together with nested-PCR permitted the detection of X. fastidiosa within sharpshooter heads with great sensitivity. It was possible to detect up to two bacteria per reaction. From 250 sharpshooter samples comprising four species (Dilobopterus costalimai, Oncometopia facialis, Bucephalogonia xanthopis and Acrogonia sp.), 87 individuals showed positive results for X. fastidiosa in a nested-PCR assay. CONCLUSIONS: The use of Chelex 100 resin allowed a fast and efficient DNA extraction to be used in the detection of X. fastidiosa in citrus plants and insect vectors by PCR and nested-PCR assays, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: The employment of efficient and sensitive methods to detect X. fastidiosa in citrus plants and insect vectors will greatly assist epidemiological studies.     

Development of a specific PCR assay for the detection of Rhizoctonia solani AG 1-IB using SCAR primers

AIMS: The aim of this study was to develop a specific and sensitive identification method for Rhizoctonia solani AG 1-IB isolates based on phylogenetic relationships of R. solani AG-1 subgroups using rDNA-internal transcribed spacer (rDNA-ITS) sequence analysis. METHODS AND RESULTS: A neighbour-joining tree analysis of 40 rDNA-ITS sequences demonstrated that R. solani AG-1 isolates cluster separately in six subgroups IA, IB, IC, ID, IE and IF. A molecular marker was generated from a random amplified polymorphic DNA fragment (RAPD). After conversion into a sequence-characterized amplified region (SCAR), a specific primer set for identification of subgroup AG 1-IB was designed for use in a polymerase chain reaction (PCR). The primer pair amplified a single DNA product of 324 bp. CONCLUSIONS: R. solani AG-1 subgroups were discriminated by sequence analysis of the ITS region. The designed SCAR primer pair allowed an unequivocal and rapid detection of R. solani AG 1-IB in plant and soil samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Sequence analysis of the rDNA-ITS region can be used for differentiation of subgroups within AG-1. The use of the developed SCAR primer set allowed a reliable and fast identification of R. solani AG 1-IB and provides a powerful tool for disease diagnosis.     

Detection of aster yellows phytoplasma in false flax based on PCR and RFLP

False flax (Camelina sativa L.) plants were found to be infected with a yellows-type disease caused by a phytoplasma in experimental plots at the Edmonton Research station. Alberta, Canada. Typical phytoplasmas were detected in the phloem cells in ultrathin sections from leaf midrib tissues examined by electron microscopy. These observations were supported by polymerase chain reaction (PCR) using two primer pairs, R16 F2n/R2 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. Aster yellows (AY) and potato witches'-broom (PWB) phytoplasma DNA samples served as controls and were used to study group relatedness. In a direct PCR assay, DNA amplification with universal primer pair R16F2n/R2 gave the expected PCR products of 1.2 kb. Based on a nested-PCR assay using the latter PCR products as templates, and a specific primer pair, R16(1)F1/R1, designed on the basis of AY phytoplasma rDNA sequences, a PCR product of 1.1 kb was obtained from each phytoplasma-infected false flax and AY sample, but not from PWB phytoplasma and healthy controls. DNA amplification with specific primer pair R16(1)F1/R1 and restriction fragment length polymorphism indicated the presence of AY phytoplasma in the infected false flax sample. This is the first reported characterization of AY phytoplasma in false flax.     

Aster Yellows Phytoplasma Identified in Scentless Chamomile by Microscopical Examinations and Molecular Characterization

Scentless chamomile (Matricaria perforata Mérat) plants were commonly found infected with a yellows-type disease caused by phytoplasma in several fields in Alberta, Canada. Typical phytoplasmas were detected in the phloem cells in ultrathin sections from leaf, stem, root and flower petiole tissues examined by electron microscopy. Application of 4′6-diamidino-2-phenylindole- 2HCl (DAPI) staining techniques confirmed the presence of the phytoplasma in these tissues. These observations were supported by polymerase chain reaction (PCR) assays, using two primer pairs, P1/P6 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. Aster yellows and potato witches′-broom (PWB) DNA phytoplasma samples served as positive controls and were used to study group relatedness. In a direct PCR assay, DNA amplification with universal primer pair P1/P6 gave the expected PCR products of 1.5 kb. Based on a nested-PCR assay using the latter PCR products, as templates, and a specific primer pair R16(1)F1/R1 designed on the basis of AY phytoplasma rDNA sequences, a PCR product of 1.1 kb was obtained from each phytoplasma-infected chamomile and AY samples but not from PWB phytoplasma and healthy chamomile controls. DNA amplification with specific primer pair R16(1)F1/R1 and restriction fragment length polymorphism indicated the presence of AY phytoplasma in the infected scentless chamomile sample.     

Development of a PCR assay to detect the potential production of nivalenol in Fusarium poae

Fusarium species can produce mycotoxins, which can contaminate cereal-based food producing adverse effects for human and animal health. In recent years, the importance of Fusarium poae has increased within the Fusarium head blight complex. Fusarium poae is known to produce trichothecenes, especially nivalenol, a potent mycotoxin able to cause a variety of toxic effects. In this study, a specific primer pair was designed based on the tri7 gene to detect potential nivalenol-producing F.?poae isolates. A total of 125 F.?poae, four F.?cerealis, two F.?culmorum, one F.?langsethiae, one F.?sporotrichioides and seven F.?graminearum, plus F.?austroamericanum, F.?meridionale, F.?graminearum sensu stricto and F.?cortaderiae from the NRRL collection were analysed, and only F.?poae isolates gave a positive result for the presence of a 296-bp partial tri7 DNA fragment. Moreover, the primer set was tested from cereal seed samples where F.?poae and other Fusarium species with a negative result for the specific reaction ( F.?graminearum, F.?oxysporum, F.?chlamydosporum, F.?sporotrichioides, F.?equiseti and F.?acuminatum) were isolated, and the expected fragment was amplified. We developed a rapid and reliable PCR assay to detect potential nivalenol-producing F.?poae isolates.     

Highly sensitive detection of Pseudomonas savastanoi pv. savastanoi in asymptomatic olive plants by nested-PCR in a single closed tube

A nested-polymerase chain reaction (PCR) has been set up to be performed in a single closed tube for the detection of Pseudomonas savastanoi pv. savastanoi. Nested-PCR coupled with dot-blot hybridization was able to detect up to one cell of the target per ml of olive extract, showing the greatest sensitivity compared with all previously reported detection assays. Validation of the developed procedure for diagnosis and epidemiological purposes was achieved by testing ca. 240 asymptomatic plant samples from olive trees. When performing the other previously reported techniques (bacterial isolation and single PCR), P. savastanoi was detected in 50 of the analyzed samples, while with the new developed nested-PCR assay, the bacterium was detected in 82 samples.     

Characterization of AfpA, an alkaline foam protein from cultures of Fusarium culmorum and its identification in infected malt

AIMS: The objective of this study was to evaluate the capability of Fusarium culmorum to produce non-hydrophobin surface-active proteins in vitro, to isolate and characterize such proteins from liquid cultures, to analyse their effect on overfoaming (gushing) of beer and to elucidate their prevalence in pure cultures and infected malt. METHODS AND RESULTS: A 20 kDa protein was isolated from liquid cultures of F. culmorum BBA 62182 upon enrichment by foaming. BLAST search with N-terminal and internal sequences of the protein revealed high homology with a hypothetical protein predicted within the F. graminearum PH1 genome sequence. Oligonucleotide primers designed to bind 30 nt upstream and downstream of the predicted gene were used to amplify a 695 nt PCR fragment from genomic DNA of F. culmorum BBA 62182. Cloning and sequencing of the product revealed a 635 nt open reading frame which had 98% homology to the predicted F. graminearium PH1 gene code. Removal of a 59 nt intron and translation resulted in a 191 amino acid protein of 20.754 kDa with a calculated pI of 9.1. Amino acids obtained by Edman sequencing of fragments within the 20 kDa protein were 100% homologous with the sequence deduced from the DNA sequence. According to its properties, the new protein was termed alkaline foam protein A (AfpA). Sequence comparison revealed some homologies with proteins in Emericella nidulans, which are involved in phialide development and response to antifungal agents. Homologies with other hypothetical fungal proteins suggest a new group of proteins, for which we suggest the name fungispumins. Addition of AfpA to beer showed that overfoaming (gushing) is not induced in stable beer but can significantly enhance this effect in beer showing moderate gushing. Use of a polyclonal anti-AfpA antibody in a Western blot revealed that the protein is produced by various F. culmorum strains and also by F. graminearum, but not by other Fusarium spp. tested. PCR testing of 69 species of Fusarium and Trichoderma reesei with a gene specific primer pair revealed that the gene may be present exclusively in F. culmorum, F. graminearum, F. cerealis, F. lunulosporum and F. oxysporum f. sp . dianthi. Immunochemical detection of AfpA in malts artificially inoculated with F. culmorum and F. graminearum showed that the protein was present in gushing inducing malts (gushing test) but absent in malts which were negative in a gushing test. CONCLUSIONS: AfpA is a member of a new protein class, fugispumins, and can be isolated from pure liquid cultures of F. culmorum. A homologous protein is synthesised by F. graminearum. The protein is produced in contaminated malt and enhances gushing of beer. The gene coding for AfpA is restricted to Fusarium species presumably involved in the induction of beer gushing. SIGNIFICANCE AND IMPACT OF THE STUDY: We describe a new class of proteins, fungispumins, the natural function of which remains to be elucidated. Findings add useful information to research on the mechanisms involved in foam stability of beer. AfpA may be useful as a marker for gushing in future quality control applications for the brewing industry.     

A new diagnostic system for ultra-sensitive and specific detection and quantification of Candidatus Liberibacter asiaticus, the bacterium associated with citrus Huanglongbing

An ultra-sensitive and quantitative diagnostic system by combining nested PCR and TaqMan® PCR in a single tube was developed for detection of “Candidatus Liberibacter asiaticus”. The procedure involves two PCR steps using the species-specific outer and inner primer pairs. Different annealing temperatures allow both the first and the second rounds of PCR to be performed sequentially in the same closed tube. The first PCR with outer primers was performed at a higher annealing temperature and with limited amount of primers to prevent interference with the inner primers during the second round of PCR. The specificity of the dual primer TaqMan® is high because the fluorescent signal can only be generated from the TaqMan® probes that are homologous to the product amplified by the outer and inner primers. This new detection system can reliably detect as few as single copies of target DNA. The sensitivity of the dual primer system is comparable to the conventional two-tube nested PCR, but it eliminates the potential risk of cross contamination commonly associated with conventional nested PCR. This one-tube dual primer TaqMan® PCR method is gel-free with reduced handling time and is cost effective. At the same time, this system provides significantly increased sensitivity, improved reliability and high through-put capability suitable for routine, large scale diagnoses of clinical plant tissue and insect samples. The technique described here is generic and can be applied to the detection of other plant pathogenic bacteria.     

Simultaneous detection of Fusarium asiaticum and Fusarium graminearum in wheat seeds using a real-time PCR method

Aims:  To develop a PCR-based method for quantitative detection of Fusarium asiaticum ( Fa ) and Fusarium graminearum ( Fg ) in wheat seeds.
Methods and Results:  Based on the sequences of the cyp51A gene, two primer pairs FaF + FaR and FgF + FgR were developed for the species-specific detection of Fa and Fg , respectively. To simultaneously detect these two phylogenetic species, a pair of primers FgaF + FgaR was developed based on the first and the second introns of β -tubulin gene. This primer pair amplified a 228-bp fragment only from Fa and Fg isolates, but not from 22 other Fusarium spp. and 13 other fungal species. A real-time PCR with this primer pair was able to quantify minute amounts of Fa and Fg DNA in wheat seeds rapidly.
Conclusions:  PCR primers designed based on the sequence of cyp51A or intron region of β -tubulin gene could allow differentiation of genetically related fungal species.
Significance and Impact of the Study:  The sensitive and quantitative detection method can be readily used in epidemiological studies and in assessing risk of Fusarium mycotoxin contamination in wheat samples.     

Use of polymerase chain reaction to detect the soft rot pathogen,Pythium myriotylum,in infected ginger rhizomes

AIMS: The aims are to establish a polymerase chain reaction (PCR)-based method for detecting Pythium myriotylum in the rhizome of ginger and diagnosing ginger soft rot and screening health seed ginger. METHODS AND RESULTS: A booster PCR method was established for detection of P. myriotylum using a specific primer selected from rDNA ITS1 region coupled with universal primer ITS2. It successfully applied to the detection of P. myriotylum in naturally infected ginger rhizomes but not from DNA of ginger rhizomes collected from field without target fungus. CONCLUSIONS: A specific method for detecting P. myriotylum was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: The new PCR method has allowed us to monitor ginger for the presence of P. myriotylum as a way of disease diagnosis or healthy seed ginger examination.     

Identification of Fusarium graminearum in cereal samples by DNA Detection Test Strips

AIMS: Development of a fast, sensitive and easy to handle method for the detection of Fusarium graminearum contamination in cereal samples by PCR. METHODS AND RESULTS: DNA Detection Test Strips were used for PCR-product detection and the method was compared to agarose gel electrophoresis. A minimum of 0.26 ng of purified target DNA was detectable with the Test Strip Detection limit in less contaminated samples was slightly lower when gel electrophoresis was used for amplicon detection. In highly contaminated samples, detection limits of both methods were similar. CONCLUSIONS: Detection of PCR products was performed in 20 min without the need of special technical equipment or hazardous fluorescent DNA dyes. SIGNIFICANCE AND IMPACT OF THE STUDY: The new method described is useful for the screening of cereals in industrial quality control.     

从粪便样品中检测动物冠状病毒的分子技术研究

目的 感染冠状病毒的动物向环境排毒主要是通过粪便,建立直接从粪便样品对动物冠状病毒进行检测的分子技术具有重要的公共卫生学意义。方法 通过计算机模拟和实验方法对已报道的2对针对冠状病毒pol基因的通用引物的通用性进行了验证。不经传统的病毒分离．直接从环境样品中提取病毒RNA,通过一步法RT-PCR进行检测,并通过分子杂交和Nested PCR扩增结合TaqMan探针实时荧光检测的PCR技术．提高对冠状病毒检测的灵敏度和准确度,并对猪、禽冠状病毒感染的临床样品进行分析检测。结果 2对引物可以覆盖所有已知的冠状病毒,包括SARS,RT-PCR产物通过测序可以确定冠状病毒种类;实时荧光定量NestedPCR有很高的灵敏度,可以灵敏地检出所有供试的阳性样品,而荧光增量实时监测可以排除凝胶电泳检查的假阳性。结论 该研究为从环境中普查和鉴定冠状病毒提供了可靠的技术方法。     

A single PCR-restriction endonuclease analysis for rapid identification of Malassezia species

AIMS: The present study describes a system based on PCR and restriction endonuclease analysis (REA) to distinguish the seven currently recognized Malassezia species. METHODS AND RESULTS: Fifty-five representative yeast isolates were examined. A single primer pair was designed to amplify the large subunit ribosomal RNA (LSU rRNA) gene of the seven Malassezia species, and identification was achieved by digestion of the PCR products with three restriction endonucleases: BanI, HaeII and MspI. A specific restriction endonuclease analysis pattern was determined for each species investigated. Moreover, PCR-REA allowed the detection and characterization of mixtures of several Malassezia species. CONCLUSION: PCR-REA of only the LSU rRNA gene is a reliable and rapid method to distinguish all Malassezia species. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR-REA represents a considerable saving in time over currently available identification procedures. This method should be evaluated on clinical material directly.     

Detection of cytomegalovirus in infant cerebrospinal fluid by conventional PCR, nested PCR and PCR-Digene

The possibility of amplification of human cytomegalovirus (HCMV) DNA in cerebrospinal fluid (CSF) for the diagnosis of HCMV central nervous system (CNS) infection in infants was studied. Single-step PCR, nested PCR and PCR-Digene were used to assay CSF specimens from 37 patients. Criteria for patient inclusion in the study were: 1. clinical manifestations suggesting CMV neuroinfection such as seizures, hypertonia, hypotonia, intracranial calcification, microcephaly, chorioretinitis; 2. any of the following symptoms: anaemia, hepetomegaly, prolonged cholestatic jaundice, or hepatitis, splenomegaly, thrombocytopenia, intrauterine hypotrophy; 3. serologic presentation, and/or positive results for CMV infection obtained by single-step PCR and PCR-Digene in urine and/or blood. PCR-Digene results were positive in 6 CSF samples. Four CSF samples were positive by nested PCR and 1 CSF sample by single step PCR. We found that the double PCR was about ten or more times more sensitive than single PCR and the PCR-Digene was only three times more sensitive than nested-PCR. The results were correlated with serology. Thirty-three out of 37 examined patients were seropositive (ELISA IgG); ELISA IgM gave positive results in 9 patients. In control studies, cells infected with other members of the herpes virus family were negative with these methods, which suggest that amplification combined with primers from the IE and the L-region of CMV is specific. In conclusion, nested-PCR seems to be the best method for early diagnosis of CMV infection in CSF due to an absence of false positive results and its high specificity and sensitivity.     

人趋化因子MIP-3α的原核可溶性表达及趋化活性分析

目的:克隆人趋化因子MIP3α,进行原核表达并初步鉴定其趋化活性。方法:从人扁桃体中提取总RNA,进行RTPCR,扩增MIP3α成熟蛋白基因,重组于pET32a(+)载体,转化大肠杆菌BL21TrxB(DE3),进行融合表达,Westernblot验证融合蛋白,金属离子亲和层析,肠激酶酶切,弱阳离子交换层析,得到纯化的MIP3α蛋白,趋化试验鉴定其趋化活性。结果:成功构建了MIP3α天然蛋白的硫氧还蛋白融合表达载体,表达并纯化出MIP3α蛋白,Westernblot证明融合蛋白能与羊抗人MIP3α抗体结合,纯化的MIP3α蛋白能趋化HEK293CCR6稳定转染细胞。结论:构建的天然MIP3α融合表达载体以可溶性蛋白的方式稳定表达MIP3α,初步纯化得到的MIP3α具有趋化HEK293CCR6稳定转染细胞的活性。