改良DAS-Dot-ELISA检测西瓜细菌性果斑病菌

以硝酸纤维素膜为载体,对Dot-ELISA法的封闭条件、包被抗体浓度、点样量等反应条件进行优化,建立改良DAS-Dot-ELISA法快速检测西瓜细菌性果斑病菌。研究发现,以含乙二胺四乙酸二钠(EDTA)的脱脂奶粉液高温处理后用于封闭,可有效降低背景;轻微振荡可提高杂交效率,减少非特异性结合。改良DAS-Dot-ELISA可快速、经济的检测西瓜果斑病菌,灵敏度达1.9×105CFU/mL。在对两批次种子样品的检测中,改良DAS-Dot-ELISA法检测带菌率分别为8.0%和6.0%,与微孔板ELISA结果完全一致;对每粒种子的检测结果,改良DAS-Dot-ELISA法与微孔板ELISA吻合率平均达99.0%,显示较好的实用前景,同时为快速检测西瓜果斑病菌提供一种新途径。     

Development of surface plasmon resonance imaging for detection of Acidovorax avenae subsp. citrulli (Aac) using specific monoclonal antibody

An immunosensor based on surface plasmon resonance imaging (SPR imaging) using a specific monoclonal antibody 11E5 (MAb 11E5) was developed for the detection of the seed-borne bacterium Acidovorax avenae subsp. citrulli (Aac), which causes fruit blotch in watermelons and cantaloupes, and compared to the conventional ELISA technique. The 1:40 mixed self-assembled monolayer (mixed SAM) surface was used for the immobilized MAb 11E5 on sensor surface for the detection of Aac. Both whole cells and broken cells of Aac were tested by using direct and sandwich detection assay. The limit of detection (LOD) of Aac using the SPR imaging technique and a direct detection assay was 10(6)cfu/ml and a subsequent amplification of the SPR signal using a polyclonal antibody (PAb) lowered the LOD to 5×10(5) cfu/ml. The LOD for the ELISA technique was 5×10(4) cfu/ml for the detection of Aac, which was slightly better than that for the SPR technique. However, the sensor surface based on SPR imaging offered a major advantage in terms of surface regeneration, allowing at least five cycles with a shorter time assay, multi-channel analysis with an application on multiplex detection, and an ease of the surface usage for the detection of Aac in the naturally infected plant. The surface was tested against the naturally infected sample and showed good selectivity toward the Aac bacteria.     

Differences in Pathogenicity between two Genetically Distinct Groups of Acidovorax avenae subsp. citrulli on Cucurbit Hosts

Using DNA fingerprinting by pulse‐field gel electrophoresis and repetitive extragenic pallindromic (REP)‐polymerase chain reaction (PCR), two distinct groups were confirmed among 64 Acidovorax avenae subsp. citrulli strains collected from a range of cucurbitaceous hosts in the USA, China, Taiwan, Thailand, Canada, Australia, Brazil and Israel. Eighty‐two percent of the group I strains were recovered from non‐watermelon hosts and the subspecies type strain was the only member of this group that utilized l ‐leucine as a sole carbon source. On the contrary, 94% of the group II strains were recovered from watermelon and 96% of them utilized l ‐leucine. Two‐week‐old watermelon cv. Crimson sweet, cantaloupe cv. Athena, pumpkin cv. Lumina and squash cv. Early yellow crookneck seedlings were susceptible to A. avenae subsp. citrulli strains representing each group with the exception of the subspecies type strain. Overall, seedlings of watermelon cv. Crimson Sweet were most susceptible to A. avenae subsp. citrulli infection followed by cantaloupe, pumpkin and squash. Group II strains were more aggressive watermelon than on other hosts. On the contrary, group I strains were moderately aggressive on all cucurbit hosts tested.     

Genome sequence of the rice-pathogenic bacterium Acidovorax avenae subsp. avenae RS-1

Acidovorax avenae subsp. avenae is a phytobacterium which is the causative agent of several plant diseases with economic significance. Here, we present the draft genome sequence of strain RS-1, which was isolated from rice shoots in a rice field in China. This strain can cause bacterial stripe of rice.     

Rapid and sensitive detection of Acidovorax citrulli in cucurbit seeds by visual loop‐mediated isothermal amplification assay

A rapid, sensitive and visual loop‐mediated isothermal amplification (LAMP) method for detecting Acidovorax citrulli in cucurbit seed was developed in this study. The LAMP primers were designed to recognize the non‐ribosomal peptide synthetase (NRPS) gene (locus tag: Aave_4658) from A. citrulli. The LAMP assay was conducted at 64°C in 1 hr with calcein as an indicator. The sensitivity and specificity of the LAMP assay were further compared with those of a conventional polymerase chain reaction (PCR). The LAMP assay is highly specific to A. citrulli, and no cross‐reaction was observed with other bacterial pathogen. The sensitivity of the LAMP assay was 100‐fold higher than that of conventional PCR with a detection limit of 1 pg of genomic DNA. Using the LAMP assay, 7 of 12 cantaloupe seedlots collected from Xinjiang province were determined to be positive for A. citrulli. In contrast, only 2 of 12 seedlots showed positive for the pathogen with conventional PCR. Moreover, A. citrulli was detected in 100% of artificially infested seedlots with 0.01% infestation or greater. Our results demonstrated that the LAMP assay was simple, visual and sensitive for detecting A. citrulli, especially in seed health testing. Hence, this method has great potential application in routine detecting seed‐borne pathogens and reducing the risk of epidemics.     

Colonization of Female Watermelon Blossoms by Acidovorax avenae ssp. citrulli and the Relationship between Blossom Inoculum Dosage and Seed Infestation

The aim of this work was to investigate the ability of Acidovorax avenae ssp. citrulli, the causal agent of bacterial fruit blotch of cucurbits (BFB), to colonize female watermelon blossoms, and to explore the relationship between blossom inoculum dosage and seed infestation. Under greenhouse conditions A. avenae ssp. citrulli colonized stigmas and styles of female watermelon blossoms reaching populations of ≈10⁷ to 10⁸ colony‐forming units (CFU) per blossom for 96 h after inoculation. Acidovorax avenae ssp. citrulli growth on stigmas was slower than that of Pseudomonas syringae Cit7, a non‐pathogenic, foliar epiphyte of tomato. While pollination reduced growth of A. avenae ssp. citrulli, but P. syringae Cit7 was unaffected. Both bacteria colonized style tissues but bacterial growth in the style was significantly less than the stigma. Blossom inoculation with ≈1 × 10³A. avenae ssp. citrulli CFU/blossom led to 36–55% infested seedlots within symptomless fruits. On average 14% of the seedlings produced from these seedlots displayed BFB symptoms. There was a strong positive correlation between A. avenae ssp. citrulli inoculum concentration applied to blossoms and the percentage of infested seedlots, as determined by the seedling grow‐out assay (R² = 0.94). However, this relationship was weaker when seedlot infestation was determined by a polymerase chain reaction‐based assay (R² = 0.34). There was also a strong positive linear relationship between A. avenae ssp. citrulli blossom inoculum dose and the mean percentage of BFB‐infected seedlings (R² = 0.99) produced in seedling grow‐out assays. These data support the hypothesis that blossom colonization might be involved in seed infestation under field conditions.     

Characterizing the mode of action of Brevibacillus laterosporus B4 for control of bacterial brown strip of rice caused by A. avenae subsp. avenae RS-1

Biological control efficacy of Brevibacillus laterosporus B4 associated with rice rhizosphere was assessed against bacterial brown stripe of rice caused by Acidovorex avenae subsp. avenae. A biochemical bactericide (chitosan) was used as positive control in this experiment. Result of in vitro analysis indicated that B. laterosporus B4 and its culture filtrates (70 %; v/v) exhibited low inhibitory effects than chitosan (5 mg/ml). However, culture suspension of B. laterosporus B4 prepared in 1 % saline solution presented significant ability to control bacterial brown stripe in vivo. Bacterization of rice seeds for 24 h yielded a greater response (71.9 %) for controlling brown stripe in vivo than chitosan (56 %). Studies on mechanisms revealed that B. laterosporus B4 suppressed the biofilm formation and severely disrupted cell membrane integrity of A. avenae subsp. avenae, causing the leakage of intracellular substances. In addition, the expression level of virulence-related genes in pathogen recovered from biocontrol-agent-treated plants showed that the genes responsible for biofilm formation, motility, niche adaptation, membrane functionality and virulence of A. avenae subsp. avenae were down-regulated by B. laterosporus B4 treatment. The biocontrol activity of B. laterosporus B4 was attributed to a substance with protein nature. This protein nature was shown by using ammonium sulfate precipitation and subsequent treatment with protease. The results obtained from this study showed the potential effectiveness of B. laterosporus B4 as biocontrol agent in control of bacterial brown stripe of rice.     

Glycosylation regulates specific induction of rice immune responses by Acidovorax avenae flagellin

Plants have a sensitive system that detects various pathogen-derived molecules to protect against infection. Flagellin, a main component of the bacterial flagellum, from the rice avirulent N1141 strain of the Gram-negative phytopathogenic bacterium Acidovorax avenae induces plant immune responses including H₂O₂ generation, whereas flagellin from the rice virulent K1 strain of A. avenae does not induce these immune responses. To clarify the molecular mechanism that leads to these differing responses between the K1 and N1141 flagellins, recombinant K1 and N1141 flagellins were generated using an Escherichia coli expression system. When cultured rice cells were treated with recombinant K1 or N1141 flagellin, both flagellins equally induced H₂O₂ generation, suggesting that post-translational modifications of the flagellins are involved in the specific induction of immune responses. Mass spectrometry analyses using glycosyltransferase-deficient mutants showed that 1,600- and 2,150-Da glycans were present on the flagellins from N1141 and K1, respectively. A deglycosylated K1 flagellin induced immune responses in the same manner as N1141 flagellin. Site-directed mutagenesis revealed that glycans were attached to four amino acid residues (Ser¹⁷⁸, Ser¹⁸³, Ser²¹², and Thr³⁵¹) in K1 flagellin. Among mutant K1 flagellins in which each glycan-attached amino acid residue was changed to alanine, S178A and S183A, K1 flagellin induced a strong immune response in cultured rice cells, indicating that the glycans at Ser¹⁷⁸ and Ser¹⁸³ in K1 flagellin prevent epitope recognition in rice.     

Rapid DNA detection by beacon-assisted detection amplification

This protocol describes a new and rapid isothermal reaction process designed to amplify and detect a specific DNA sequence in purified DNA extracted from cultured cells. The protocol uses a DNA nanomachine that comprises two molecular switches that function in concert to isothermally amplify and detect a DNA target. First, a molecular beacon detection switch is 'activated' only if a DNA target sequence is present. A DNA primer and DNA polymerase are used to lock the beacon in an activated conformation. Second, an amplification and signal-transduction switch is initiated following successful activation. A nicking endonuclease and the DNA polymerase are used to replicate the DNA target. Both switches operate simultaneously at 40 °C in a single reaction to rapidly generate multiple copies of the DNA target in a cyclic polymerization reaction. This protocol enables femtomole amounts of a DNA target to be reproducibly amplified and detected in <40 min. We demonstrate the successful use of this protocol in assays containing synthetic DNA components and purified DNA extracted from biological samples.     

Rapid Discrimination between Groups I and II of Acidovorax citrulli Using a Primer Pair Specific to a pilL Gene

Acidovorax citrulli can be divided into two genetic groups: group I and group II based primarily on pulsed‐field gel electrophoresis (PFGE) and multilocus sequence classification (MLST). To distinguish more rapidly between strains of the two groups, a pair of specific primer for specific polymerase chain reaction (PCR) that can identify group II strains was designed based on the pilL gene of a group II strain, AAC00‐1. PCR results showed that a 332‐bp band was generated for 51 of 52 group II strains whereas only three of 93 group I strains were positive, largely consisting with previous studies of A. citrulli classification. Results of PCR showed the primers were able to detect group II strains of A. citrulli and distinguish between strains of groups I and II rapidly and accurately.     

Preliminary Evidence for Phytoalexin Production by Eucalyptus urophylla Leaves as a Response to Inoculation with the Incompatible Pathogen Acidovorax avenae pv. avenae

Leaves of eucalyptus ( Eucalyptus urophylla ) were infiltrated with a cell suspension of the incompatible pathogen Acidovorax avenae pv. avenae and showed a typical hypersensitive response within 24h. Necrotic leaf areas were excised, vacuum infiltrated with 40% ethanol and left under continuous agitation at room temperature for 24h. The diffusate was concentrated, partitioned with ethyl-acetate, concentrated to dryness and resuspended in a small volume of methanol. The biological activity of extracts was evaluated by an agar diffusion method against noncompatible bacteria ( A. avenae pv. avenae and Xanthomonas axonopodis pv. manihotis ) and fungi ( Penicillium sp. and Aspergillus sp. ). Inhibition haloes, when present, were always larger in extracts from leaves infiltrated with the incompatible bacterium than the water control. Thin layer chromatography resolution of crude extracts from leaves infiltrated with both incompatible pathogen cell suspension and water, followed by bioautography with Thieleviopsis paradoxa, consistently rendered, in both situations, a large, diffuse inhibition halo near the origin, assumed to be due to preformed antimicrobial substances. However, extracts from leaves infiltrated with the living cells of the incompatible pathogen gave rise to a smaller, second inhibition halo, near the front, that was interpreted as being one or several phytoalexins.     

Rapid real-time PCR assay for detection and quantitation of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk

Using fluorescence resonance energy transfer technology and Lightcycler analysis, we developed a real-time PCR assay with primers and probes designed by using IS900 which allowed rapid detection of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk. Initially, the PCR parameters (including primer and probe levels, assay volume, Mg(2+) concentration, and annealing temperature) were optimized. Subsequently, the quantitative ability of the assay was tested and was found to be accurate over a broad linear range (3 x 10(6) to 3 x 10(1) copies). The assay sensitivity when purified DNA was used was determined to be as low as five copies, with excellent reproducibility. A range of DNA isolation strategies was developed for isolating M. avium subsp. paratuberculosis DNA from spiked milk, the most effective of which involved the use of 50 mM Tris HCl, 10 mM EDTA, 2% Triton X-100, 4 M guanidinium isothiocyante, and 0.3 M sodium acetate combined with boiling, physical grinding, and nucleic acid spin columns. When this technique was used in conjunction with the real-time PCR assay, it was possible to consistently detect <100 organisms per ml of milk (equivalent to 2,000 organisms per 25 ml). Furthermore, the entire procedure (extraction and PCR) was performed in less than 3 h and was successfully adapted to quantify M. avium subsp. paratuberculosis in spiked milk from heavily and mildly contaminated samples.     

Rapid detection of maize DNA sequence variation.

The allele-specific polymerase chain reaction (ASPCR) has been used to determine the genotype of maize lines at two loci, wx and NPI288. The ASPCR method uses allele-specific oligonucleotide primers in PCR amplifications to amplify and discriminate simultaneously between polymorphic alleles. The success of this technique relies on the specific failure of PCR to amplify with primers that do not perfectly match the DNA sequence of one of the allelic variants. Amplification results were evaluated by dot-blot hybridization using an alkaline-phosphatase-coupled probe. The technique's speed, accuracy, sensitivity, and high throughput make it valuable for plant-breeding applications.     

Specific detection of Leuconostoc mesenteroides subsp. mesenteroides with DNA primers identified by randomly amplified polymorphic DNA analysis

Randomly amplified polymorphic DNA analysis using primer 239 (5' CTGAAGCGGA 3') was performed to characterize Leuconostoc sp. strains. All the strains of Leuconostoc mesenteroides subsp. mesenteroides (with the exception of two strains), two strains formerly identified as L. gelidum, and one strain of Leuconostoc showed a common band at about 1.1 kb. This DNA fragment was cloned and sequenced in order to verify its suitability for identifying L. mesenteroides subsp. mesenteroides strains.     

Rapid DNA detection by interface PCR on nanoparticles

A novel, rapid DNA detection method based on fluorescence quenching of quantum dots (QDs) by gold nanoparticles (GNPs) through polymerase chain reaction (PCR) was developed. In proof-of-concept experiments, the length of the amplicon DNA ranging from 152 to 1003 base pairs (bp) could be determined based on quenched fluorescence intensity with 136 bp as the lower limit of effective range. And the real sample detections were also achieved successfully by this developed method. Therefore, this DNA detection method has the potential to be the powerful gene diagnostic tool.     

Rapid DNA mapping by fluorescent single molecule detection

DNA mapping is an important analytical tool in genomic sequencing, medical diagnostics and pathogen identification. Here we report an optical DNA mapping strategy based on direct imaging of individual DNA molecules and localization of multiple sequence motifs on the molecules. Individual genomic DNA molecules were labeled with fluorescent dyes at specific sequence motifs by the action of nicking endonuclease followed by the incorporation of dye terminators with DNA polymerase. The labeled DNA molecules were then stretched into linear form on a modified glass surface and imaged using total internal reflection fluorescence (TIRF) microscopy. By determining the positions of the fluorescent labels with respect to the DNA backbone, the distribution of the sequence motif recognized by the nicking endonuclease can be established with good accuracy, in a manner similar to reading a barcode. With this approach, we constructed a specific sequence motif map of lambda-DNA. We further demonstrated the capability of this approach to rapidly type a human adenovirus and several strains of human rhinovirus.     

Rapid detection of Chattonella marina by PCR combined with dot lateral flow strip

Journal of Applied Phycology - In this study a novel technique referred to as PCR combined with dot lateral flow strip (PCDS) is proposed and its application to the detection of harmful microalgae...