Amplification of Nucleic Acids by Polymerase Chain Reaction (PCR) and Other Methods and their Applications

DNA corresponding to 16S rDNA and the 16S-23S rDNA intergenic spacer (ITS) from 22 reference strains of acetic acid bacteria, representing the diversity of the family Acetobacteraceae, and 24 indigenous acetic acid bacteria isolated from wine fermentations were analysed by PCR-RFLP. Frateuria aurantia LMG 1558 T and Escherichia coli ATCC 11775 T were included as outgroups. PCRamplified products of about 1450 bp were obtained from the 16S rDNA of all the strains and products of between 675 and 800 bp were obtained from the 16S-23S rDNA ITS. PCR products were digested with 4-base-cutting restriction enzymes in order to evaluate the degree of polymorphism existing among these strains. Of the enzymes tested, TaqI and RsaI were the most discriminatory and showed no intraspecific variations in the restriction patterns. Restriction analysis of the 16S rDNA with these enzymes is proposed as a rapid and reliable method to identify acetic acid bacteria at the level of genus and species (or related species group) and its applicability to identification of indigenous acetic acid bacteria was demonstrated. The same degree of distinction as that for the 16S rDNA analysis was obtained within reference strains of acetic acid bacteria by PCR-RFLP of the 16S-23S rDNA ITS. However, 16S-23S rDNA ITS restriction patterns of strains isolated from wine did not match those of any of the reference strains. Thus, PCR-RFLP of the 16S-23S rDNA ITS is not a useful method to identify isolates of acetic acid bacteria at the species level, although it may be an adequate method to detect intraspecific differentiation.

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“…The design of the primers was carried out by following criteria described previously (Bej et al, 1991).…”

Section: Methodsmentioning

confidence: 99%

Identification of acetic acid bacteria by RFLP of PCR-amplified 16S rDNA and 16S-23S rDNA intergenic spacer.

Ruiz¹,

Poblet²,

Mas³

et al. 2000

International Journal of Systematic and Evolutionary Microbiology

122

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“…The nucleic acid sequencebased amplification assay, also known as self-sustained sequence replication (3SR), can be performed under isothermal conditions and is useful for detection of clinically relevant pathogens (21,47). PCR is the prototype nucleic acid amplification method, and it has been extensively evaluated (6,69,70,115). This technique has evolved from a laborious and relatively insensitive assay into an extremely sensitive and highly flexible procedure.…”

Section: Genotypingmentioning

confidence: 99%

DNA fingerprinting of medically important microorganisms by use of PCR

1994

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Selected segments of any DNA molecule can be amplified exponentially by PCR. This technique provides a powerful tool to detect and identify minimal numbers of microorganisms. PCR is applicable both in diagnosis and in epidemiology. By amplification of hypervariable DNA domains, differences can be detected even among closely related strains. PCR fingerprinting is a valuable tool for medical microbiologists, epidemiologists, and microbial taxonomists. The current state of PCR-mediated genotyping is reviewed, and a comparison with conventional molecular typing methods is included. Because of its speed and versatility, PCR fingerprinting will play an important role in microbial genetics, epidemiology, and systematics.

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“…Single-strand, antisense DNA probes were generated by PCR using the pBS-AaSvp as the template and were labeled with [ -32 P]dATP (Bej et al 1991). The specific activity of the probe was determined by scintillation counting, and it was added to the hybridization buffer at a concentration of 1 10 6 dpm/ml solution.…”

Section: Northern Blot Hybridizationmentioning

confidence: 99%

A COUP-TF/Svp homolog is highly expressed during vitellogenesis in the mosquito Aedes aegypti

Miura¹,

Zhu²,

Dittmer³

et al. 2002

Journal of Molecular Endocrinology

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In the mosquito Aedes aegypti, vitellogenesis is activated via an ecdysteroid hormonal cascade initiated by a blood meal. The functional ecdysone receptor is a heterodimer composed of the ecdysone receptor (EcR) and ultraspiracle, the homolog of the retinoid X receptor. The precise tuning of this hormonal response requires participation of both positive and negative transcriptional regulators. In Drosophila, Svp, a homolog of chicken ovalbumin upstream promoter transcription factor (COUP-TF), inhibits ecdysone receptor complex-mediated transactivation in vitro and in vivo. Here we report the cloning and characterization of the Svp homolog in mosquito Aedes aegypti, AaSvp. It possesses a high degree of amino acid sequence similarity to the members of the COUP-TF/Svp subfamily. AaSvp transcripts and protein are present in the fat body at high levels from the state of arrest to about 60 h post blood meal. AaSvp binds strongly to a variety of direct repeats of the sequence AGGTCA, but weakly to inverted repeats such as hsp27EcRE. Transient transfection assays in Drosophila S2 cells showed that AaSvp was able to repress 20-hydroxyecdysone (20E)-dependent transactivation mediated by the mosquito ecdysteroid receptor complex. These data suggest that AaSvp negatively regulates the 20E signaling in the fat body during mosquito vitellogenesis.

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Amplification of Nucleic Acids by Polymerase Chain Reaction (PCR) and Other Methods and their Applications

Cited by 139 publications

References 195 publications

Identification of acetic acid bacteria by RFLP of PCR-amplified 16S rDNA and 16S-23S rDNA intergenic spacer.

Identification of acetic acid bacteria by RFLP of PCR-amplified 16S rDNA and 16S-23S rDNA intergenic spacer.

DNA fingerprinting of medically important microorganisms by use of PCR

A COUP-TF/Svp homolog is highly expressed during vitellogenesis in the mosquito Aedes aegypti

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