Isolation of a Pseudomonas solanacearum-specific DNA probe by subtraction hybridization and construction of species-specific oligonucleotide primers for sensitive detection by the polymerase chain reaction.

A subtraction hybridization technique was employed to make a library enriched for Pseudomonas solanacearum-specific sequences. One cloned fragment, PS2096, hybridized under stringent conditions to DNA of 82 P. solanacearum strains representing all subgroups of the species. Other plant-associated bacteria, including closely related species such as Pseudomonas capacia, Pseudomonas picketti, or Pseudomonas syzygii, did not hybridize to PS2096. A minimum number of between 4 x 10(5) and 4 x 10(6) P. solanacearum cells could routinely be detected with PS2096 labelled either with [32P]dCTP or with digoxigenin-11-dUTP. To improve the sensitivity of detection, PS2096 was sequenced to allow the construction of specific oligonucleotide primers to be used for polymerase chain reaction (PCR) amplification. After 50 cycles of amplification, 5 to 116 cells, depending on the strain, could reproducibly be detected by visualization of a 148-bp PCR product on an agarose gel. A preliminary field trial in Burundi with the probe and PCR primers has confirmed that they are sensitive tools for specifically detecting low-level infections of P. solanacearum in potato tubers.     

Use of tRNA consensus primers to indicate subgroups of Pseudomonas solanacearum by polymerase chain reaction amplification.

Polymerase chain reaction amplification of DNA from 112 Pseudomonas solanacearum strains with the tRNA consensus primers T3A and T5A divided the species into three fingerprint groups. These groups correspond well with previous divisions made by restriction fragment length polymorphism analysis. This polymerase chain reaction test is a facile method for rapidly classifying P. solanacearum strains.     

Specific and sensitive detection of Ralstonia solanacearum in soil on the basis of PCR amplification of fliC fragments

Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops. A specific and sensitive PCR detection method that uses primers targeting the gene coding for the flagella subunit, fliC, was established. Based on the first fliC gene sequence of R. solanacearum strain K60 available at GenBank, the Ral_fliC PCR primer system was designed; this system yielded a single 724-bp product with the DNAs of all of the R. solanacearum strains tested. However, R. pickettii and four environmental Ralstonia isolates also yielded amplicons. The Ral_fliC PCR products obtained with 12 strains (R. solanacearum, R. pickettii, and environmental isolates) were sequenced. By sequence alignment, Rsol_fliC primers specific for R. solanacearum were designed. With this primer system, a specific 400-bp PCR product was obtained from all 82 strains of R. solanacearum tested. Six strains of R. pickettii and several closely related environmental isolates yielded no PCR product; however, a product was obtained with one Pseudomonas syzygii strain. A GC-clamped 400-bp fliC product could be separated in denaturing gradient gels and allowed us to distinguish P. syzygii from R. solanacearum. The Rsol_fliC PCR system was applied to detect R. solanacearum in soil. PCR amplification, followed by Southern blot hybridization, allowed us to detect about one target DNA molecule per PCR, which is equivalent to 10(3) CFU g of bulk soil(-1). The system was applied to survey soils from different geographic origins for the presence of R. solanacearum.     

Polymerase chain reaction amplification of naphthalene-catabolic and 16S rRNA gene sequences from indigenous sediment bacteria.

We report the amplification of bacterial genes from uninoculated surface and subsurface sediments by the polymerase chain reaction (PCR). PCR amplification of indigenous bacterial 16S ribosomal DNA genes was unsuccessful when subsurface sediment containing approximately 10(7) cells.g-1 was added directly to a PCR mixture. However, when 10 mg of sediment was inoculated with approximately 10(5) cells of Pseudomonas putida G7, the nahAc naphthalene dioxygenase gene characteristic of the P. putida G7 NAH7 plasmid was detected by PCR amplification. Southern blotting of the PCR amplification product improved sensitivity to 10(3) to 10(4) cells from samples inoculated with P. putida G7, but controls with no sediment added showed that the PCR was partially inhibited by the sediments. Lysozyme-sodium dodecyl sulfate-freeze-thaw DNA extraction was combined with gel electrophoretic partial purification in the presence of polyvinylpyrrolidone to render DNA from indigenous bacteria in surface or subsurface sediment samples amplifiable by PCR using eubacterial 16S ribosomal DNA primers. The nahAc gene could also be amplified from indigenous bacteria by using nahAc-specific primers when PCR conditions were modified by increasing Taq and primer concentrations. Restriction digests of the nahAc amplification products from surface and subsurface sediments revealed polymorphism relative to P. putida G7. The procedures for DNA extraction, purification, and PCR amplification described here demonstrate that the PCR is a potentially useful tool in studies of function- and taxon-specific DNA from indigenous microbial communities in sediment and groundwater environments.     

Detection by in vitro amplification of the alpha-toxin (phospholipase C) gene from Clostridium perfringens

A polymerase chain reaction (PCR) with thermostable DNA polymerase from Thermus aquaticus is described for the specific amplification of the phospholipase C (alpha-toxin) gene of Clostridium perfringens. A set of primers selected for their high specificity could detect Cl. perfringens in stools with a detection limit of approximately 5 x 10² bacteria, after bi-amplification. A modified PCR without thermal steps was performed to rapidly amplify, with a yield of 60%, the DNA template. With this PCR method Cl. perfringens alpha-toxin gene could be detected within 2 h. The PCR method detected alpha-toxin positive Cl. perfringens but did not react with phospholipase C-producing Bacillus cereus, Pseudomonas aeruginosa, Cl. sordellii and Cl. bifermentans.
The amplified PCR products were screened through ethidium bromide agarose gel electrophoresis or, in only 1 h, with the PhastSystem (Pharmacia). This PCR satisfies the criteria of specificity, sensitivity and rapidity required for a useful tool in epidemiology and for the diagnosis of the pathogen Cl. perfringens as it may be used directly on stool samples.     

A dose response for radiation-induced intrachromosomal DNA rearrangements detected by inverse polymerase chain reaction.

X-ray-induced intrachromosomal DNA rearrangements were detected in the 5' region of the MYC gene of cells of the human bladder carcinoma cell line, EJ-30, by using PCR with inverted primers. When the cells were allowed to repair/misrepair for 6 or 23 h after irradiation, the frequency of rearrangements increased with dose from (0.7 +/- 0.4) x 10(-5) per copy of MYC for unirradiated cells to (3.2 +/- 0.7) x 10(-5) after 30 Gy, (5.4 +/- 1.2) x 10(-5) after 70 Gy, and (5.9 +/- 1.0) x 10(-5) after 100 Gy. No significant difference was observed between 6 and 23 h of repair. Sequences obtained from the products suggest that there was no homology between the two sequences involved in the recombination event and that there was no clustering of breakpoints. The procedure is relatively simple, requiring only one digestion with a rare-cutting restriction enzyme prior to PCR amplification of the DNA purified from irradiated cells. The site of enzyme digestion is located between a pair of primer sites 120 bp apart for which the primers face in opposite directions. If no intrachromosomal rearrangement has occurred, no PCR product would be obtained. However, if an intrachromosomal rearrangement has occurred between two regions located on either side of the primer sites, an episome or duplication event would result if the rearrangement had occurred either within the same chromatid or between two sister chromatids, respectively. Digestion between the primers would linearize an episome or release a linear molecule containing the duplicated primer sites from a larger molecule. After both types of rearrangement events, the primers would be facing each other and would be located on either end of the linear molecule; and if they are less than approximately 5 kb apart, PCR amplification should result in a product. This procedure is relatively simple and rapid and does not require any cell division after irradiation or phenotypic selection of mutants. Also, quantification is based on the number of PCR products detected in a known amount of DNA, and not on a precise determination of the amount of PCR amplification that has occurred. Thus the inverse PCR procedure has the potential ofbeing used as an assay to detect variations in radiation-induced frequencies of DNA rearrangements.     

Detection of Pseudomonas aeruginosa from clinical and environmental samples by amplification of the exotoxin A gene using PCR.

PCR was used to detect Pseudomonas aeruginosa from water samples by amplifying a 396-bp region of the exotoxin A (ETA) structural gene sequence. The identify of the amplified 396-bp fragment was confirmed by digesting it with PvuI restriction endonuclease, which produced the predicted 246- and 150-bp fragments. Specific primers amplified ETA-positive P. aeruginosa DNA, whereas other species of Pseudomonas and GC-rich bacteria did not yield any 396-bp fragment. The specificity and sensitivity of the assay were 100 and 96%, respectively, which confirms the assay's reliability for diagnostic and epidemiological studies. The assay can detect as few as 5 to 10 cells in a 10-ml water sample or 0.1 pg of P. aeruginosa DNA per reaction mixture (5 microliters) by ethidium bromide staining of an agarose gel. Ten-times-lower concentrations were detected by hybridization with a digoxigenin-labeled oligonucleotide probe internal to the PCR product. With this PCR method, ETA-positive P. aeruginosa was detected in animal cage water samples at a level of 40 cells per ml. This method is rapid and less cumbersome than other diagnostic methods for the identification of P. aeruginosa strains. The method described can be used to detect a low level of P. aeruginosa from environmental and clinical samples without the use of selective media or additional biochemical tests.     

Detection of Pseudomonas savastanoi pv. savastanoi in olive plants by enrichment and PCR

The sequence of the gene iaaL of Pseudomonas savastanoi EW2009 was used to design primers for PCR amplification. The iaaL-derived primers directed the amplification of a 454-bp fragment from genomic DNA isolated from 70 strains of P. savastanoi, whereas genomic DNA from 93 non-P. savastanoi isolates did not yield this amplified product. A previous bacterial enrichment in the semiselective liquid medium PVF-1 improved the PCR sensitivity level, allowing detection of 10 to 100 CFU/ml of plant extract. P. savastanoi was detected by the developed enrichment-PCR method in knots from different varieties of inoculated and naturally infected olive trees. Moreover, P. savastanoi was detected in symptomless stem tissues from naturally infected olive plants. This enrichment-PCR method is more sensitive and less cumbersome than the conventional isolation methods for detection of P. savastanoi.     

Use of ethidium bromide monoazide for quantification of viable and dead mixed bacterial flora from fish fillets by polymerase chain reaction

Ethidium bromide monoazide (EMA) was utilized to selectively allow conventional PCR amplification of target DNA from viable but not dead cells from a broth culture of bacterial mixed flora derived from cod fillets. The universal primers designated DG74 and RW01 that amplify a 370-bp sequence of a highly conserved region of all eubacterial 16S rDNA were used for the PCR. The use of 10 microg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The minimum amount of EMA to completely inhibit the PCR amplification of DNA derived from dead bacterial cells was 0.8 microg/ml. Amplification of target DNA from only viable cells in a suspension with dead cells was selectively accomplished by first treating the cells with 1 microg/ml of EMA. A standard curve was generated relating the intensity of fluorescence of DNA bands to the log of CFU of mixed bacterial cultures for rapidly assessing the number of genomic targets per PCR derived from the number of CFU. A linear range of DNA amplification was exhibited from 1 x 10(2) to 1 x 10(5) genomic targets per PCR. The viable/dead cell discrimination with the EMA-PCR method was evaluated by comparison with plate counts following freezing and thawing. Thawing frozen cell suspensions initially containing 1 x 10(5) CFU/ml at 4, 20, and 37 degrees C yielded a 0.8 log reduction in the number of viable cells determined by both plate counts and EMA-PCR. In contrast, thawing for 5 min at 70 degrees C resulted in a 5 log reduction in CFU derived from plate counts (no CFU detected) whereas the EMA-PCR procedure resulted in only a 2.8 log reduction in genomic targets, possibly reflecting greater damage to enzymes or ribosomes at 70 degrees C to a minority of the mixed population compared to membrane damage.     

Detection and quantification of copper-denitrifying bacteria by quantitative competitive PCR

We developed a quantitative competitive PCR (QC-PCR) system to detect and quantify copper-denitrifying bacteria in environmental samples. The primers were specific to copper-dependent nitrite reductase gene (nirK). We were able to detect about 200 copeis of nirK in the presence of abundant non-specific target DNA and about 1.2 x 10(3)Pseudomonas sp. G-179 cells from one gram of sterilized soil by PCR amplification. A 312-bp nirK internal standard (IS) was constructed, which showed very similar amplification efficiency with the target nirKfragment (349 bp) over 4 orders of magnitude (10(3)-10(6)). The accuracy of this system was evaluated by quantifying various known amount of nirK DNA. The linear regressions were obtained with a R(2) of 0.9867 for 10(3)copies of nirK, 0.9917 for 10(4) copies of nirK, 0.9899 for 10(5) copies of nirK and 0.9846 for 10(6) copies of nirK. A high correlation between measured nirK and calculated nirK (slope of 1.0398, R(2)=0.9992) demonstrated that an accurate measurement could be achieved with this system. Using this method, we quantified nirK in several A-horizon and stream sediment samples from eastern Tennessee. In general, the abundance of nirK was in the range of 10(8)-10(9) copies g soil(-1) dry weight. The nirK content in the soil samples appeared correlated with NH(4)(N) content in the soil. The activities of copper-denitrifying bacteria were evaluated by quantifying cDNA of nirK. In most of sample examined, the content of nirK cDNA was less than 10(5) copies g soil(-1) dry weight. Higher nirK cDNA content (>10(6) copies g soil(-1) dry weight) was detected from both sediment samples at Rattlebox Creek and the Walker Branch West Ridge. Although the stream sediment samples at the Walker Branch West Ridge contained less half of the nirK gene content as compared to A-horizon sample, the activities of copper-denitrifying bacteria were almost 600 times higher than in the A-horizon sample.     

Detection of polychlorinated biphenyl degradation genes in polluted sediments by direct DNA extraction and polymerase chain reaction.

It was the aim of this study to specifically detect the DNA sequences for the bphC gene, the meta-cleavage enzyme of the aerobic catabolic pathway for biphenyl and polychlorinated biphenyl degradation, in aquatic sediments without prior cultivation of microorganisms by using extraction of total DNA, PCR amplification of bphC sequences, and detection with specific gene probes. The direct DNA extraction protocol used was modified to enhance lysis efficiency. Crude extracts of DNA were further purified by gel filtration, which yielded DNA that could be used for the PCR. PCR primers were designed for conserved regions of the bphC gene from a sequence alignment of five known sequences. The specificity of PCR amplification was verified by using digoxigenin-labeled DNA probes which were located internal to the amplified gene sequence. The detection limit for the bphC gene of Pseudomonas paucimobilis Q1 and Pseudomonas sp. strain LB400 was 100 cells per g (wet weight) or approximately five copies of the target sequence per PCR reaction mixture. In total-DNA extracts of aerobic top layers of sediment samples obtained from three different sampling sites along the Elbe River, which has a long history of anthropogenic pollution, Pseudomonas sp. strain LB 400-like sequences for the bphC gene were detected, but P. paucimobilis Q1 sequences were not detected. No bphC sequences were detected in an unpolluted lake sediment. A restriction analysis did not reveal any heterogeneity in the PCR product, and the possibility that sequences highly related to the bphC gene (namely, nahC and todE) were present was excluded.(ABSTRACT TRUNCATED AT 250 WORDS)     

荧光假单胞菌Tn5转座诱变及对青枯病生防特性

目的：利用Tn5转座诱变荧光假单胞菌PF20001,研究所获得的突变株对青枯病的生防效果。方法：利用三亲本杂交方式,将带有转座子Tn5的Tn5-102（含luxAB）的质粒pTR102成功地转入PF20001,利用平板相互拮抗法分析突变株对青枯病致病菌的拮抗作用。结果：通过诱导Tn5转座,得到荧光假单胞菌PF20001的Tn5插入突变库。经平板相互拮抗实验发现,菌株PF20001-lux-48拮抗圈明显大于野生型（半径达0.35cm）。用Tn5-lux特异引物进行PCR扩增,结果显示只有以该突变株的DNA为模板才能得到300bp的扩增产物,证实该菌株基因组中有Tn5插入。结论：Tn5的插入使菌株PF20001对青枯病生物防治能力增强。     

基于EMA-qPCR的茄科青枯菌活体检测技术的建立

【目的】利用特异性核酸染料叠氮溴乙锭(Ethidium monoazide bromide, EMA)与实时荧光定量PCR技术相结合, 建立一种能有效区分青枯菌死活细胞的检测方法。【方法】样品DNA制备前经EMA渗透预处理, 再进行实时荧光定量PCR特异扩增菌体DNA。【结果】终浓度为2.0 mg/L的EMA能有效排除1.0×107 CFU/mL灭活青枯菌细胞DNA的扩增, 对活细胞和不可培养状态(Viable but non-culturable, VBNC)活菌的DNA扩增均没有影响。当每个定量PCR反应体系中的活细胞在5.0×100?5.0×104 CFU范围内时, 扩增Ct值与定量PCR反应体系中活细胞CFU对数值呈良好的负相关性(R2=0.992 5)。比较EMA-qPCR法和平板计数法对经过不同温度短期保存的青枯菌检测结果发现, 待检样品可在24 °C与4 °C冷藏条件下短期保存。【结论】本研究建立的EMA-qPCR方法能有效检测青枯菌VBNC细胞和有效区分死活菌, 避免或减少青枯菌PCR检测的假阳性和假阴性。     

Evaluation of a rapid microbial detection method via phage lytic amplification assay coupled with Live/Dead fluorochromic stains

AIMS: To develop a method for rapid detection of bacteria via bacteriophage amplification coupled with exogenous fluorochromic stains. METHODS AND RESULTS: A method for the rapid detection of bacteria was developed which consisted of exposing the sample suspected to contain target cells to host-specific phage. After at least one infection cycle, bacteria known to be infected by the phage (helper cells) were added and the number of nascent phage particles was estimated using the Live/Dead BacLight Bacterial Viability kit. Using Pseudomonas aeruginosa, it was shown that the dead helper cell population following phage infection was proportional to the initial number of target cells present in the original sample. Approximately 1 x 10(1) CFU per ml of P. aeruginosa could be detected within 4 h without the need for enrichment. CONCLUSIONS: The phage lytic amplification assay coupled with exogenous fluorochromic stains was able to detect approx. 1 x 10(1) CFU per ml of the target bacterium within 4 h. SIGNIFICANCE AND IMPACT OF THE STUDY: A method to detect low number of bacterial cells in a sample within 4 h without the need for enrichment was developed.     

Detection of Pseudomonas savastanoi pv. savastanoi in Olive Plants by Enrichment and PCR

The sequence of the gene iaaL of Pseudomonas savastanoi EW2009 was used to design primers for PCR amplification. The iaaL-derived primers directed the amplification of a 454-bp fragment from genomic DNA isolated from 70 strains of P. savastanoi, whereas genomic DNA from 93 non-P. savastanoi isolates did not yield this amplified product. A previous bacterial enrichment in the semiselective liquid medium PVF-1 improved the PCR sensitivity level, allowing detection of 10 to 100 CFU/ml of plant extract. P. savastanoi was detected by the developed enrichment-PCR method in knots from different varieties of inoculated and naturally infected olive trees. Moreover, P. savastanoi was detected in symptomless stem tissues from naturally infected olive plants. This enrichment-PCR method is more sensitive and less cumbersome than the conventional isolation methods for detection of P. savastanoi.     

白菜的EST标记及其对油菜的通用性

根据白菜的表达序列标签,设计了28对引物。在对引物、dNTP、MgCl2的浓度及退火温度等参数进行测试后,建立了合适的PCR反应体系。在此反应体系下,以构建EST的白菜自交系A的DNA为模板,对设计的引物进行了筛选,发现有18对引物能对白菜DNA扩增出产物。用筛选出来的引物分别对17个白菜类品种进行PCR扩增,用琼脂糖凝胶电泳分析其产物的多态性,发现10对引物有多态性,这占了筛选引物的55.6%。为检测白菜EST标记的通用性,进一步利用设计的引物对不同油菜品种的DNA进行PCR扩增。在检测的28对引物中,共有24对引物能扩增出产物,占引物总数的85.7%,显示多态性的引物为18对,占引物总数的64.3%.。在对白菜DNA能扩增出产物的18对引物中,对油菜完全可用,且有13对引物产生多态性。而在那些对白菜未扩增出产物的10对引物中,也有6对能扩增出产物,其中5对显示多态性。文章研究结果证明,通过EST建立分子标记是可行的,而且这种标记对近缘物种是可通用的。     

Rapid Detection of Phytophthora nicotianae in Infected Tobacco Tissues and Soil Samples Based on Its Ypt1 Gene

This paper describes the development of a polymerase chain reaction (PCR) assay for the detection of Phytophthora nicotianae , the causal agent of Phytophthora blight of tobacco and other plants. The PCR primers were designed based on a Ras-related protein ( Ypt 1) gene, and 115 isolates representing 26 species of Phytophthora and 29 fungal species of plant pathogens were used to test the specificity of the primers. PCR amplification with species-specific (Pn) primers resulted in a product of 389 bp only from isolates of P. nicotianae . The detection sensitivity with Pn primers was 1 ng of genomic DNA. Using Ypt 1F/ Ypt 1R as first-round amplification primers, followed by a second round using the primer pair Pn1/Pn2, a nested PCR procedure was developed, which increased the detection sensitivity 100-fold to 10 pg. PCR with the Pn primers could also be used to detect P. nicotianae from naturally infected tobacco tissues and soil. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management.     

Occurrence of the strA-strB streptomycin resistance genes in Pseudomonas species isolated from kiwifruit plants

The occurrence of strA-strB streptomycin-resistance genes within transposon Tn5393 was examined in Pseudomonas syringae pv. actinidiae, P. syringae pv. syringae, and P. marginalis, isolated from kiwifruit plants in Korea and Japan. PCR amplification with primers specific to strA-strB revealed that three of the tested Pseudomonas species harbored these genes for a streptomycin-resistance determinant. Tn5393, containing strA-strB, was also identified with PCR primers designed to amplify parts of tnpA, res, and tnpR. No IS elements were detected within tnpR, nor were they found in the intergenic region between tnpR and strA. Nucleotide sequence analysis indicated that the strA sequence of P. syringae pv. actinidiae contained a single nucleotide alteration at position 593 (CAA-->CGA), as compared to Tn5393a in P. syringae pv. syringae. This resulted in an amino acid change, from Gln to Arg.     

PCR-based specific detection of Ralstonia solanacearum by amplification of cytochrome c1 signal peptide sequences

A polymerase chain reaction (PCR)-based method was developed to detect the DNA of Ralstonia solanacearum, the causal agent of bacterial wilt in various crop plants. One pair of primers (RALSF and RALSR), designed using cytochrome c1 signal peptide sequences specific to R. solanacearum, produced a PCR product of 932 bp from 13 isolates of R. solanacearum from several countries. The primer specificity was then tested using DNA from 21 isolates of Ralstonia, Pseudomonas, Burkholderia, Xanthomonas, and Fusarium oxysporum f. sp. dianthi. The specificity of the cytochrome c1 signal peptide sequences in R. solanacearum was further confirmed by a DNA-dot blot analysis. Moreover, the primer pair was able to detect the pathogen in artificially inoculated soil and tomato plants. Therefore, the present results indicate that the primer pair can be effectively used for the detection of R. solanacearum in soil and host plants.     

Multiplex PCR assay for detection of four major bacterial pathogens causing rainbow trout disease

A multiplex PCR (mPCR) method was designed for the simultaneous detection of 4 major fish pathogens, Flavobacterium psychrophilum, Lactococcus garvieae, Pseudomonas aeruginosa, and P. putida. Each of the 4 pairs of oligonucleotide primers exclusively amplified the 16S rDNA gene of their targeted microorganism. The average detection limits for each organism amplified by mPCR were 2 colony-forming units (CFU) of F. psychrophilum, 3 CFU of L. garvieae, 3 CFU of P. aeruginosa, and 5 CFU of P. putida in mixed cultures. Multiplex PCR did not produce any nonspecific amplification products when tested against 28 related species of bacteria. High amounts of DNA from 1 bacterial species had a significant effect on the amplification sensitivity of the other bacterial species when these were present in lower concentrations in the multiplex reaction. The mPCR assay proved useful for the detection of the bacteria in naturally infected fish. The assay is a sensitive, specific, and reproducible diagnostic tool for the simultaneous detection of 4 pathogenic bacteria that cause disease in fish and offers a potentially useful alternative to the conventional culture-based method.