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1.
Erica D. Dawson Amber W. Taylor James A. Smagala Kathy L. Rowlen 《Molecular biotechnology》2009,42(1):117-127
We developed molecular diagnostic assays for the detection of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subsp. equisimilis (SDSE), two streptococcal pathogens known to cause both pharyngitis and more invasive forms of disease in humans. Two real-time
PCR assays coupled with an internal control were designed to be performed in parallel. One assay utilizes a gene target specific
to GAS, and the other utilizes a gene target common to the two species. Both assays showed 2–3 orders of magnitude improved
analytical sensitivity when compared to a commercially available rapid antigen test. In addition, when compared to standard
culture in an analysis of 96 throat swabs, the real-time PCR assays resulted in clinical sensitivity and specificity of 91.7
and 100%, respectively. As capital equipment costs for real-time PCR can be prohibitive in smaller laboratories, the real-time
PCR assays were converted to a low-density microarray format designed to function with an inexpensive photopolymerization-based
non-enzymatic signal amplification (NESA™) method. S. pyogenes was successfully detected on the low-density microarray in less than 4 h from sample extraction through detection. 相似文献
2.
Salmonella causes the majority of infections in humans and homeothermic animals. This article describes a specific polymerase chain
reaction (PCR) method developed for a rapid identification of Salmonella. A gyrB-targeted species-specific primer pair, S-P-for (5′-GGT GGT TTC CGT AAA AGT A-3′) and S-P-rev (5′-GAA TCG CCT GGT TCT TGC-3′),
was successfully designed. PCR with all the Salmonella strains produced a 366- bp DNA fragment that was absent from all the non-Salmonella strains tested. The detection limit of the PCR was 0.01 ng with genomic DNA or 3.2 cells per assay. Good specificity was
also demonstrated by fecal samples, from which only the gyrB gene of Salmonella was amplified. Using the culture-PCR method, 27 isolates on Salmonella-Shigella (SS) medium were rapidly identified as Salmonella, which was confirmed by the sequencing of the gyrB gene. 相似文献
3.
The aim of this study is to develop a polymerase chain reaction (PCR) assay for rapid detection of Cupriavidus metallidurans. PCR primers targeting the Signal transduction histidine kinase gene were designed and designated Cm-F1/Cm-R1. Strains of C. metallidurans were positively identified. The size of the PCR products was 437 bp, as expected. This PCR method enables monitoring of industrial,
environmental and clinical sources for presence of C. metallidurans. 相似文献
4.
Guma MK Abdeldaim Kristoffer Strålin Jens Korsgaard Jonas Blomberg Christina Welinder-Olsson Björn Herrmann 《BMC microbiology》2010,10(1):310
Background
Streptococcus pneumoniae and Haemophilus influenzae cause pneumonia and as Neisseria meningitidis they are important agents of meningitis. Although several PCR methods have been described for these bacteria the specificity is an underestimated problem. Here we present a quantitative multiplex real-time PCR (qmPCR) for detection of S. pneumoniae (9802 gene fragment), H. influenzae (omp P6 gene) and N. meningitidis (ctrA gene). The method was evaluated on bronchoalveolar lavage (BAL) samples from 156 adults with lower respiratory tract infection (LRTI) and 31 controls, and on 87 cerebrospinal fluid (CSF) samples from meningitis patients. 相似文献5.
Vibrio harveyi, pathogenic to fish, harbor a hemolysin gene vhh, the homologues of which are found in many species of the Genus Vibrio. In this study, we investigated the prevalence of vhh gene among V. harveyi isolated from Penaeus monodon hatcheries in India by polymerase chain reaction (PCR). The vhh was detected in 67 of the 70 V. harveyi isolates tested in this study using different combinations of PCR primers. A variant vhh gene detected in a minority of strains was cloned, sequenced, and the recombinant protein was expressed in Escherichia coli. The deduced amino acid sequence of the cloned gene was 86% similar to the previously reported amino acid sequences of VHH.
The results of this study suggest that though V. harveyi strains invariably harbor vhh, the sequence variants of the hemolysin gene exist that may impede their detection by PCR. 相似文献
6.
Torres-Calzada C Tapia-Tussell R Quijano-Ramayo A Martin-Mex R Rojas-Herrera R Higuera-Ciapara I Perez-Brito D 《Molecular biotechnology》2011,49(1):48-55
Colletotrichum capsici is an important fungal species that causes anthracnose in many genera of plants causing severe economic losses worldwide.
A primer set was designed based on the sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for
use in a conventional PCR assay. The primer set (CcapF/CcapR) amplified a single product of 394 bp with DNA extracted from
20 Mexican isolates of C. capsici. The specificity of primers was confirmed by the absence of amplified product with DNA of four other Colletotrichum species and eleven different fungal genera. This primer set is capable of amplifying only C. capsici from different contaminated tissues or fungal structures, thereby facilitating rapid diagnoses as there is no need to isolate
and cultivate the fungus in order to identify it. The sensitivity of detection with this PCR method was 10 pg of genomic DNA
from the pathogen. This is the first report of a C. capsici-specific primer set. It allows rapid pathogen detection and provides growers with a powerful tool for a rational selection
of fungicides to control anthracnose in different crops and in the post-harvest stage. 相似文献
7.
Ferreira LQ Avelar KE Vieira JM de Paula GR Colombo AP Domingues RM Ferreira MC 《Current microbiology》2007,54(5):348-353
The Bacteroides genus, the most prevalent anaerobic bacteria of the intestinal tract, carries a plethora of the mobile elements, such as
plasmids and conjugative and mobilizable transposons, which are probably responsible for the spreading of resistance genes.
Production of β-lactamases is the most important resistance mechanism including cephalosporin resistance to β-lactam agents
in species of the Bacteroides fragilis group. In our previous study, the cfxA gene was detected in B. distasonis species, which encodes a clinically significant broad-spectrum β-lactamase responsible for widespread resistance to cefoxitin
and other β-lactams. Such gene has been associated with the mobilizable transposon Tn4555. Therefore, the aim of this study was to detect the association between the cfxA gene and the presence of transposon Tn4555 in 53 Bacteroides strains isolated in Rio de Janeiro, Brazil, by PCR assay. The cfxA gene was detected in 11 strains and the Tn4555 in 15. The transposon sequence revealed similarities of approximately 96% with the B. vulgatus sequence which has been deposited in GenBank. Hybridization assay was performed in attempt to detect the cfxA gene in the transposon. It was possible to associate the cfxA gene in 11 of 15 strains that harbored Tn4555. Among such strains, 9 presented the cfxA gene as well as Tn4555, but in 2 strains the cfxA gene was not detected by PCR assay. Our results confirm the involvement of Tn4555 in spreading the cfxA gene in Bacteroides species. 相似文献
8.
Chai-Hoon Khoo Yoke-Kqueen Cheah Learn-Han Lee Jiun-Horng Sim Noorzaleha Awang Salleh Shiran Mohd Sidik Son Radu Sabrina Sukardi 《Antonie van Leeuwenhoek》2009,96(4):441-457
The increased occurrence of Salmonella occurrence in local indigenous vegetables and poultry meat can be a potential health hazards. This study is aimed to detect
the prevalence of twenty different virulence factors among Salmonella enterica strains isolated from poultry and local indigenous vegetables in Malaysia via an optimized, rapid and specific multiplex
PCR assay. The assay encompasses a total of 19 Salmonella pathogenicity islands genes and a quorum sensing gene (sdiA) in three multiplex reaction sets. A total of 114 Salmonella
enterica isolates belonging to 38 different serovars were tested. Each isolate in under this study was found to possess up to 70%
of the virulence genes tested and exhibited variable pathogenicity gene patterns. Reproducibility of the multiplex PCR assay
was found to be 100% and the detection limit of the optimized multiplex PCR was tested with lowest detectable concentration
of DNA 0.8 pg μl−1. This study demonstrated various Salmonella pathogenicity island virulence gene patterns even within the same serovar. This sets of multiplex PCR system provide a fast
and reliable typing approach based on Salmonella pathogenicity islands, thus enabling an effective monitoring of emerging pathogenic Salmonella strains as an additional tool in Salmonella surveillance studies. 相似文献
9.
Loop-mediated isothermal amplification (LAMP) is a promising nucleic acid assay for rapid and cost-effective detection of
pathogen-specific sequences within a sample. Development of an appropriate taxonomic group-specific LAMP assay highly relies
on the design of proper primers to cover all major members of the taxon. Regarding this fact, we designed and evaluated a
new LAMP primer set specific to prt (rfbS) gene for rapid identification of Salmonella serogroup D serotypes. Unlike the previously reported LAMP assay for serogroup D which detects solely the non-typhoidal serotypes;
the new LAMP primers set detects both typhoidal and non-typhoidal serotypes of this serogroup with a detection limit of 10
CFU/rection. Furthermore, the technique was successfully applied to artificially contaminated meat samples with an inoculation
level of 1–5 CFU/250 ml of Salmonella Enteritidis, following a 5-h pre-enrichment step in tryptic soy broth. Overall, the new LAMP assay and its optimized setup
would be useful for fast diagnosis of food poisoning incidents caused by these bacteria. 相似文献
10.
X Liu J Fang M Zhang X Wang W Wang Y Gong X Xi M Li 《World journal of microbiology & biotechnology》2012,28(3):1013-1020
Contamination of Cronobacter spp. (Enterobacter sakazakii) in infant formulas and other food products is a severe problem. Here a loop-mediated isothermal amplification (LAMP) assay
was developed for rapidly detecting Cronobacter spp. in powdered infant formula. Sequences of 16S/23S rDNA internal intergenic spacer of Cronobacter spp. were used as the target template to design LAMP primers. The detection outcome can be evaluated by the white precipitate
or the fluorescence intensity under ultraviolet irradiation, both visible to naked eyes. The sensitivity and specificity of
the LAMP assay was further analyzed in comparison with that of regular PCR and real time quantitative PCR. The results showed
that all of Cronobacter spp. strains display positive reaction to the detections while all of the non-Cronobacter spp. strains were negative, and that the LAMP assay exhibits a high sensitivity of 9.1 fg/μL (The sensitivity of regular
PCR and real time quantitative PCR is 91 and 9.1 pg/μL, respectively.). The amplified reaction could be accomplished in about
1 h, with the results visible to naked eyes. Hence, the LAMP assay developed by this study can provide a rapid and simple
approach for the detection of Cronobacter spp. in infant formula. 相似文献
11.
X. Zhou Z. Wang K. Jiang Y. Wei J. Lin X. Sun K. Tang 《Applied Biochemistry and Microbiology》2007,43(4):439-443
A total of 38 endophytic fungus strains were isolated from Taxus chinensis var. mairei by the aseptic technique. Genomic DNA was extracted from isolated endophytic fungi and subjected to polymerase chain reaction
(PCR) analysis for the presence of the Taxus taxadiene synthase (TS) gene, a rate-limiting enzyme gene in the taxol biosynthetic pathway. Twelve out of 38 isolated endophytic fungus strains
showed PCR positive for the ts gene. Subsequently, taxol and its related compounds were extracted from culture filtrates and mycelia of the PCR positive
strains, separated by column chromatography, and analyzed by High Performance Liquid Chromatography and Mass Spectrum. The
analysis result showed that 3 strains could produce taxol and its related compounds at the detectible level. This study indicates
that molecular detection of the ts gene is an efficient method for primary screening of taxol or its related compound-producing endophytic fungi, which can
improve prominently screening efficiency.
Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2007, Vol. 43, No. 4, pp. 490–494.
The text was submitted by the authors in English. 相似文献
12.
Sina M Coldewey Maike Hartmann Dorothea S Schmidt Uta Engelking Sya N Ukena Florian Gunzer 《BMC microbiology》2007,7(1):21
Background
Enterohemorrhagic E. coli (EHEC), a subgroup of Shiga toxin (Stx) producing E. coli (STEC), may cause severe enteritis and hemolytic uremic syndrome (HUS) and is transmitted orally via contaminated foods or from person to person. The infectious dose is known to be very low, which requires most of the bacteria to survive the gastric acid barrier. Acid resistance therefore is an important mechanism of EHEC virulence. It should also be a relevant characteristic of E. coli strains used for therapeutic purposes such as the probiotic E. coli Nissle 1917 (EcN). In E. coli and related enteric bacteria it has been extensively demonstrated, that the alternative sigma factor σS, encoded by the rpoS gene, acts as a master regulator mediating resistance to various environmental stress factors. 相似文献13.
Zarizal Suhaili Saiful Azmi Johari Mastura Mohtar Ahmad Rushdi Tan Abdullah Affandi Ahmad Abdul Manaf Ali 《World journal of microbiology & biotechnology》2009,25(2):253-258
The aim of this study was to develop a methicillin-resistant Staphylococcus aureus (MRSA) detection method based on the melting temperature analysis profiling of S. aureus clinical isolates from three different hospitals in Malaysia. Simplex and duplex real-time PCR assay was used for the simultaneous
detection of nuc (species-specific) and mecA (methicillin-resistance) genes in a single SYBR Green I real-time PCR tube assay. Evaluations were based on the melting temperature
(T
m) analysis of the amplicons using 23 S. aureus clinical isolates including three ATCC S. aureus standard strains. Real-time PCR amplification products with melting peaks at 78.39 ± 0.4°C and 74.41 ± 0.6°C were detected
for nuc and mecA genes, respectively. Each real-time PCR assay was completed within two hours. This rapid genotypic method is useful for the
detection of resistant determinant (mecA) and identification of S. aureus (nuc) clinical isolates, thus benefiting patient therapy in hospitals. 相似文献
14.
A. V. Loskutov G.-Q. Song K. C. Sink 《In vitro cellular & developmental biology. Plant》2008,44(4):239-245
Callus selection (CS) and the flamingo-bill explant (FB) methods were evaluated for efficacy in transformation for celery.
Agrobacterium tumefaciens strains EHA105 and GV3101, each with the bar gene under the promoters NOS (pGPTV-BAR) or 35S (pDHB321.1), were used. Leaf explants were inoculated and co-cultivated for
2 d in the dark. Calluses emerged on the explants on callus medium (C), Murashige and Skoog (MS) medium + 2,4-Dichlorophenoxyacetic
acid (2,4-D) (2.3 μM) + kinetin (2.8 μM) + timentin (300 mg·l−1). Calluses 4- to 6-wk-old were selected for glufosinate (GS) resistance by a two step method. First, calluses were transferred
to C medium + GS 0.35, 0.5, 1, 2, 5, or 10 mg·l−1; calluses formed only with 0, 0.35 and 0.5 mg·l−1 GS. All growing calluses from 0 and 0.35 mg·l−1 and a few from 0.5 mg·l−1, were divided and placed back on C + GS 0.35–0.5 mg·l−1 for another 5–6 wk. Second, tolerant clones were again divided and placed on C + GS 1–50 mg·l−1. When cultivar XP85 was inoculated with both strains, using pGPTVBAR, 19 glufosinate resistant (GR) callus clones were selected,
but shoots regenerated only for strain EHA105 inoculations. When both of the strains (each with pDHB321.1) were inoculated
on cv. XP166, 3 and 12 GR calluses occurred for EHA105 and GV3101, respectively. Using CS, a total of 34 GR callus clones
were selected, and shoots were regenerated from over 50% of them on Gamborg B5 medium + 6-(γ, γ-dimethylallylamino) purine
2ip (4.9 μM) + naphthaleneacetic acid (NAA; 1.6 μM) and rooted on MS in 5–6 mo total time. Conversely, using FB with inoculation
by GV3101/pDHB321.1 on cv. XP166 yielded putative transgenic celery plants confirmed by polymerase chain reaction (PCR) in
just 6 wk. Transformation of the bar gene into celery was confirmed by PCR for 5 and 6 CS and FB lines, respectively. Southern blot analyses indicated 1–2 copies
in CS lines and 1 copy in FB lines. Herbicide assays on whole plants with 100 and 300 mg·l−1 glufosinate indicated a range of low to high tolerance for lines derived by both methods. The bar gene was found to be Mendelian inherited in one self-fertile CS derived line. 相似文献
15.
Attacin, a 20 kDa antibacterial peptide, plays an important role in immunity. To understand this gene better, gene cloning,
expression and biological activity detection of Attacin A was carried out in present study. The full-length open reading frame
(ORF) coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template. The gene was inserted directionally into the prokaryotic expression vector pET-32a (+). The resulting recombinant
plasmid was transformed into E. coli
Rosetta. SDS–PAGE was carried out to detect the expression product which was induced by IPTG. The antimicrobial activity and hemolysis
activity were tested in vitro after purification. Agarose gel electrophoresis indicated that the complete ORF of Attacin A
gene has been cloned successfully from Drosophila stimulated by E. coli which includes 666 bp and encodes 221 AA. The gene encoding mature Attacin A protein was amplified by PCR from the recombinant
plasmid containing Attacin A, which includes 570 bp in all. SDS–PAGE analysis demonstrated that the fusion protein expressed
was approximately 39.2 kDa. Biological activities detection showed that this peptide exhibited certain antibacterial activity
to several G− bacteria, as well as minor hemolysis activity for porcine red blood cells. In conclusion, Attacin A gene was
cloned and expressed successfully. It was the basis for further study of Attacin. 相似文献
16.
Background
Shiga toxin-producing Escherichia coli (STEC), including E. coli O157:H7, are responsible for numerous foodborne outbreaks annually worldwide. E. coli O157:H7, as well as pathogenic non-O157:H7 STECs, can cause life-threating complications, such as bloody diarrhea (hemolytic colitis) and hemolytic-uremic syndrome (HUS). Previously, we developed a real-time PCR assay to detect E. coli O157:H7 in foods by targeting a unique putative fimbriae protein Z3276. To extend the detection spectrum of the assay, we report a multiplex real-time PCR assay to specifically detect E. coli O157:H7 and screen for non-O157 STEC by targeting Z3276 and Shiga toxin genes (stx1 and stx2). Also, an internal amplification control (IAC) was incorporated into the assay to monitor the amplification efficiency.Methods
The multiplex real-time PCR assay was developed using the Life Technology ABI 7500 System platform and the standard chemistry. The optimal amplification mixture of the assay contains 12.5 μl of 2 × Universal Master Mix (Life Technology), 200 nM forward and reverse primers, appropriate concentrations of four probes [(Z3276 (80 nM), stx1 (80 nM), stx2 (20 nM), and IAC (40 nM)], 2 μl of template DNA, and water (to make up to 25 μl in total volume). The amplification conditions of the assay were set as follows: activation of TaqMan at 95 °C for 10 min, then 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 60 s.Results
The multiplex assay was optimized for amplification conditions. The limit of detection (LOD) for the multiplex assay was determined to be 200 fg of bacterial DNA, which is equivalent to 40 CFU per reaction which is similar to the LOD generated in single targeted PCRs. Inclusivity and exclusivity determinants were performed with 196 bacterial strains. All E. coli O157:H7 (n = 135) were detected as positive and all STEC strains (n = 33) were positive for stx1, or stx2, or stx1 and stx2 (Table 1). No cross reactivity was detected with Salmonella enterica, Shigella strains, or any other pathogenic strains tested.Conclusions
A multiplex real-time PCR assay that can rapidly and simultaneously detect E. coli O157:H7 and screen for non-O157 STEC strains has been developed and assessed for efficacy. The inclusivity and exclusivity tests demonstrated high sensitivity and specificity of the multiplex real-time PCR assay. In addition, this multiplex assay was shown to be effective for the detection of E. coli O157:H7 from two common food matrices, beef and spinach, and may be applied for detection of E. coli O157:H7 and screening for non-O157 STEC strains from other food matrices as well.17.
18.
Aguilar-Tipacamú G Rosario-Cruz R Miller RJ Guerrero FD Rodriguez-Vivas RI García-Vázquez Z 《Experimental & applied acarology》2011,54(3):301-311
Dialelic crosses and backcrosses of pyrethroid resistant (RR) and susceptible (SS) Rhipicephalus (Boophilus) microplus tick strains were carried out and the substitution (Phe-Ile) within the sodium channel gene was monitored in order to analyze
the effects of the genotype on the pyrethroid resistance phenotype as measured by the larval packet test (LPT). Parental strains:
susceptible (SS) and resistant (RR); dialelic crosses: RS (♂RR × ♀SS), and SR (♂SS × ♀RR); and backcrosses: RS × SS, RS × RR,
SR × SS and SR × RR were infested on 280 kg calves. Resistance type (monogenic or polygenic) and effective dominance were
determined based on the discriminant concentration (DC) for cipermethrine (0.5%), deltamethrine (0.09%) and flumethrine (0.01%).
Allele specific PCR (AS-PCR) was used for genotyping, looking at a sodium channel mutation (Phe-Ile substitution). The mortality
rates and allele frequency of susceptible and pyrethroid resistant reference strains were 0% mortality and 90% RR alleles
for resistant strain, and 100% mortality and 0% RR alleles as measured by the larval packet test (LPT) and allele specific
PCR (AS-PCR) respectively. Backcrossed strain SR × RR showed an effective dominance (DML) of 0.605 for cypermethrin, 0.639 for deltamethrin and 0.498 for flumethrin, while survival of backcrosses RS × SS, RS × RR
and SR × SS showed a significant tendency to recesivity. Backcrossed strain SR × RR (69.4%) also showed a higher RR genotype
frequency with regards to RS × SS (25.5%), RS × RR (36.7%) and SR × SS (32.0%), however, susceptible allele was inherited
in general as an incomplete dominant trait. Monogenic inheritance hypothesis was tested and the results showed monogenic inheritance
for cypermethrin and flumethrin (P < 0.05) but not for deltamethrin (P > 0.05). However, significant correlation was found between RR genotype and the survival rate for all three pyrethroids used
(P < 0.05), suggesting that a single substitution on the sodium channel gene can be responsible for resistance to pyrethroids
as a class, due to the high frequency for RR genotypes. Combination with different mutations or metabolic resistance mechanisms
cannot be excluded. 相似文献
19.
20.
The invasive aspergillosis, which is commonly caused by Aspergillus fumigatus (A. fumigatus), has increased in recent years. Traditional methods for finding out antifungal resistant strains would take more than 2 weeks,
which cannot satisfy the needs of rapid detection. In this study, a real-time PCR method for detection of the serial itraconazole-resistant
strains of A. fumigatus isolated from a lung aspergilloma patient was developed. The results showed that the TacMAN-MGB probes, which were covered
the loci Gly54, Leu98, Gly138, and Met220 of the enzyme CYP51A coded by the gene cyp51A, as well as the 34-bp tandem repeated sequence in the promoter region (−288 and −322 from the start codon) of this gene,
could detect the serial itraconazole-resistant strains of A. fumigatus in our study. Besides, this method takes just 6 h to complete the whole detection. 相似文献