Molecular Methods for the Detection and Identification of Pseudomonas stutzeri in Pure Culture and Environmental Samples

ABSTRACT Global poliovirus surveillance involves virus isolation from stool and environmental samples, intratypic differential (ITD) by PCR, and sequencing of the VP1 region to distinguish vaccine (Sabin), vaccine-derived, and wild-type polioviruses and to ensure an appropriate response. This cell culture algorithm takes 2 to 3 weeks on average between sample receipt and sequencing. Direct detection of viral RNA using PCR allows faster detection but has traditionally faced challenges related to poor sensitivity and difficulties in sequencing common samples containing poliovirus and enterovirus mixtures. We present a nested PCR and nanopore sequencing protocol that allows rapid (<3 days) and sensitive direct detection and sequencing of polioviruses in stool and environmental samples. We developed barcoded primers and a real-time analysis platform that generate accurate VP1 consensus sequences from multiplexed samples. The sensitivity and specificity of our protocol compared with those of cell culture were 90.9% (95% confidence interval, 75.7% to 98.1%) and 99.2% (95.5% to 100.0%) for wild-type 1 poliovirus, 92.5% (79.6% to 98.4%) and 98.7% (95.4% to 99.8%) for vaccine and vaccine-derived serotype 2 poliovirus, and 88.3% (81.2% to 93.5%) and 93.2% (88.6% to 96.3%) for Sabin 1 and 3 poliovirus alone or in mixtures when tested on 155 stool samples in Pakistan. Variant analysis of sequencing reads also allowed the identification of polioviruses and enteroviruses in artificial mixtures and was able to distinguish complex mixtures of polioviruses in environmental samples. The median identity of consensus nanopore sequences with Sanger or Illumina sequences from the same samples was >99.9%. This novel method shows promise as a faster and safer alternative to cell culture for the detection and real-time sequencing of polioviruses in stool and environmental samples.

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Multiplication Characteristics of FRNA Phage and Its Utility as an Indicator for Pathogenic Viruses

Water Science & Technology ◽

10.2166/wst.1993.0335 ◽

1993 ◽

Vol 27 (3-4) ◽

pp. 133-136 ◽

Cited By ~ 4

Author(s):

Naoyuki Kamiko ◽

Shinichiro Ohgaki

Keyword(s):

Bacterial Growth ◽

Growth Phase ◽

Substrate Concentration ◽

Pure Culture ◽

Environmental Samples ◽

Cultivation Temperature ◽

Pathogenic Viruses ◽

Phage Multiplication ◽

Rna Phage

The possibility of multiplication of F-specific RNA phage (FRNA phage) multiplication in the environment was investigated. Using the Qβ strain in pure culture as a model FRNA phage, the effects of bacterial growth phase, substrate concentration and cultivation temperature on phage multiplication were studied. Similar experiments with environmental samples from raw sewage were then preformed.

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Detection and identification of poliovirus in environmental samples using nucleic acid hybridization

Canadian Journal of Microbiology ◽

10.1139/m90-113 ◽

1990 ◽

Vol 36 (9) ◽

pp. 664-669 ◽

Cited By ~ 2

Author(s):

David R. Preston ◽

G. Rasul Chaudhry ◽

Samuel R. Farrah

Keyword(s):

Tissue Culture ◽

Nucleic Acid ◽

Environmental Samples ◽

Nucleic Acid Hybridization ◽

Dot Blot ◽

Polio Virus ◽

Detection And Identification ◽

Specificity And Sensitivity ◽

Hybridization Procedure ◽

Great Specificity

A procedure was developed to effectively extract viral RNA from poliovirus tissue-culture lysates while eliminating the hybridization background associated with tissue cultures uninfected with poliovirus. Poliovirus cDNA cloned into a pUC vector was used as probe. Both the recombinant plasmids and the cDNA showed great specificity towards poliovirus. However, both probes hybridized with the single-stranded DNA coliphage [Formula: see text]. Tissue culture was found to be an effective method to increase the number of viruses found in environmental samples to a level detectable by hybridization procedures, whereas direct hybridization of RNA from unamplified and highly concentrated raw wastewater showed poor hybridization signals. The specificity and sensitivity of the hybridization procedure developed during these studies indicate that this method may be best suited for the identification rather than the detection of viruses isolated from environmental samples. Key words: nucleic acid hybridization, polio virus, water, dot blot.

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Rapid extraction of lipid biomarkers from pure culture and environmental samples using pressurized accelerated hot solvent extraction

Journal of Microbiological Methods ◽

10.1016/s0167-7012(97)00081-x ◽

1997 ◽

Vol 31 (1-2) ◽

pp. 19-27 ◽

Cited By ~ 62

Author(s):

Sarah J Macnaughton ◽

Tonya L Jenkins ◽

Michael H Wimpee ◽

Misti R Cormiér ◽

David C White

Keyword(s):

Solvent Extraction ◽

Pure Culture ◽

Environmental Samples ◽

Lipid Biomarkers ◽

Rapid Extraction

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Moving towards an integrated approach to molecular detection and identification ofToxoplasma gondii

Parasitology ◽

10.1017/s0031182009991065 ◽

2009 ◽

Vol 137 (1) ◽

pp. 1-11 ◽

Cited By ~ 329

Author(s):

C. SU ◽

E. K. SHWAB ◽

P. ZHOU ◽

X. Q. ZHU ◽

J. P. DUBEY

Keyword(s):

Molecular Detection ◽

Genetic Characterization ◽

Integrated Approach ◽

Epidemiological Studies ◽

Molecular Methods ◽

Maximum Information ◽

The Past ◽

Phylogenetic Studies ◽

Detection And Identification

SUMMARYThe development of simple, sensitive and rapid methods for the detection and identification ofToxoplasma gondiiis important for the diagnosis and epidemiological studies of the zoonotic disease toxoplasmosis. In the past 2 decades, molecular methods based on a variety of genetic markers have been developed, each with its advantages and limitations. The application of these methods has generated invaluable information to enhance our understanding of the epidemiology, population genetics and phylogeny ofT. gondii. However, since most studies focused solely on the detection but not genetic characterization ofT. gondii, the information obtained was limited. In this review, we discuss some widely used molecular methods and propose an integrated approach for the detection and identification ofT. gondii, in order to generate maximum information for epidemiological, population and phylogenetic studies of this key pathogen.

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Molecular Detection and Identification of Brettanomyces/Dekkera bruxellensis and Brettanomyces/Dekkera anomalus in Spoiled Wines

Applied and Environmental Microbiology ◽

10.1128/aem.70.3.1347-1355.2004 ◽

2004 ◽

Vol 70 (3) ◽

pp. 1347-1355 ◽

Cited By ~ 65

Author(s):

Luca Cocolin ◽

Kalliopi Rantsiou ◽

Lucilla Iacumin ◽

Roberto Zironi ◽

Giuseppe Comi

Keyword(s):

Direct Detection ◽

Molecular Methods ◽

Enzyme Analysis ◽

Restriction Enzyme Analysis ◽

Rrna Gene ◽

Brettanomyces Bruxellensis ◽

Dna And Rna ◽

Detection And Identification ◽

26S Rrna ◽

Total Agreement

ABSTRACT In this paper we describe the development of a PCR protocol to specifically detect Brettanomyces bruxellensis and B. anomalus. Primers DB90F and DB394R, targeting the D1-D2 loop of the 26S rRNA gene, were able to produce amplicons only when the DNA from these two species were used. No amplification product was obtained when DNA from other Brettanomyces spp. or wine yeasts were used as the templates. The 305-bp product was subjected to restriction enzyme analysis with DdeI to differentiate between B. bruxellensis and B. anomalus, and each species could be identified on the basis of the different restriction profiles. After optimization of the method by using strains from international collections, wine isolates were tested with the method proposed. Total agreement between traditional identification and molecular identification was observed. The protocol developed was also used for direct detection of B. bruxellensis and B. anomalus in wines suspected to be spoiled by Brettanomyces spp. Application of culture-based and molecular methods led us to the conclusion that 8 of 12 samples were spoiled by B. bruxellensis. Results based on the application of molecular methods suggested that two of the eight positive samples had been infected more recently, since specific signals were obtained at both the DNA and RNA levels.

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