Comparison of DNA extraction from cervical cells collected in PreservCyt solution for the amplification of <i>Chlamydia trachomatis</i>

Keegan, Helen; Boland, Clara; Malkin, Alison; Griffin, Mairead; Ryan, Fergus; Lambkin, Helen

doi:10.1111/j.1365-2303.2005.00239.x

Cited by 15 publications

(19 citation statements)

References 39 publications

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“…The SurePath TM fixative should diminish (if not eliminate) proteolytic degradation. Fixative solutions may crosslink proteins and nucleic acids, so as to interfere with proteolytic enzymes and potentially inhibit cellular lysis [33,34]. For our purposes of MS-based proteomics, the “fixative” attribute of the SurePath TM preservative fluid proved to be advantageous.…”

Section: Discussionmentioning

confidence: 99%

A feasibility study to identify proteins in the residual Pap test fluid of women with normal cytology by mass spectrometry-based proteomics

et al. 2014

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BackgroundThe proteomic analysis of body fluids is a growing technology for the identification of protein biomarkers of disease. Given that Papanicolaou tests (Pap tests) are routinely performed on over 30 million women annually in the U.S. to screen for cervical cancer, we examined the residual Pap test fluid as a source of protein for analysis by mass spectrometry (MS). In the liquid-based Pap test, cervical cells are collected from the ectocervix and placed into an alcohol-based fixative prior to staining and pathologic examination. We hypothesized that proteins shed by cells of the female genital tract can be detected in the Pap test fixative by MS-based proteomic techniques. We examined the feasibility of using residual fluid from discarded Pap tests with cytologically “normal” results to optimize sample preparation for MS analysis. The protein composition of the cell-free Pap test fluid was determined by silver staining of sodium dodecyl sulfate -polyacrylamide gels, and the abundance of serum proteins was examined by Western immunoblot using an antibody against human serum albumin. Both pooled and individual samples were trypsin digested and analyzed by two-dimensional MS/MS. Proteins were identified by searching against the Human Uniprot database, and characterized for localization, function and relative abundance.ResultsThe average volume of the residual Pap test fluid was 1.5 ml and the average protein concentration was 0.14 mg/ml. By Western immunoblot we showed that the amount of albumin in each sample was significantly reduced compared to normal serum. By MS/MS, we identified 714 unique proteins in pooled Pap test samples and an average of 431 proteins in individual samples. About 40% of the proteins identified were extracellular or localized to the plasma membrane. Almost 20% of the proteins identified were involved in immunity and defense, characteristic of the healthy cervical-vaginal proteome. By merging the protein sets from the individual and pooled Pap test samples, we created a “Normal Pap test Core Proteome” consisting of 153 proteins.ConclusionsResidual Pap test fluid contains a sufficient amount of protein for analysis by MS and represents a valuable biospecimen source for the identification of protein biomarkers for gynecological diseases.

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Section: Discussionmentioning

confidence: 99%

A feasibility study to identify proteins in the residual Pap test fluid of women with normal cytology by mass spectrometry-based proteomics

et al. 2014

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“…BD SurePath and Hologic PreservCyt media are both alcohol-based preservatives and are designed to fix cellular and subcellular components and aid in the cytological evaluation of the obtained smears. Fixative solutions can prove problematic for downstream molecular extraction methods because of the potential to inhibit cellular lysis, because of the potential to interfere with the proteolytic enzymes used in this process, or due to cross-linking of nucleic acids and proteins (5,6). Some prior studies have reported poor recovery of nucleic acids from SurePath and PreservCyt media and inferred that the DNA had degraded over time during storage (7-10).…”

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confidence: 99%

Long-Term Stability of Human Genomic and Human Papillomavirus DNA Stored in BD SurePath and Hologic PreservCyt Liquid-Based Cytology Media

et al. 2013

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We evaluated the effect of storage at 2 to 8°C on the stability of human genomic and human papillomavirus (HPV) DNA stored in BD SurePath and Hologic PreservCyt liquid-based cytology media. DNA retained the ability to be extracted and PCR amplified for more than 2.5 years in both medium types. Prior inability to detect DNA in archived specimens may have been due to failure of the extraction method to isolate DNA from fixed cells. Liquid-based cytology (LBC) media (BD SurePath [Becton, Dickinson and Company] and Hologic PreservCyt [Hologic Inc.]) are routinely used to prepare liquid Pap preparations for cervical cancer screening. In the United States, molecular human papillomavirus (HPV) testing out of the LBC vial is recommended (i) as a triage test for determining whether a woman with atypical squamous cells of undetermined significance (ASC-US) needs immediate colposcopic evaluation and (ii) as an adjunct to cervical cytology, "cotesting," for cervical cancer screening of women of age 30 years and older. In addition, there is a growing interest in the use of LBC media for primary HPV screening in which positive specimens could be reflexed to cytology, HPV type-specific genotyping, or another triage method, although molecular tests have yet to be approved for this intended use (1, 2). LBC media were originally developed and approved for the preservation and preparation of cells for Pap smear evaluation (3, 4). BD SurePath and Hologic PreservCyt media are both alcohol-based preservatives and are designed to fix cellular and subcellular components and aid in the cytological evaluation of the obtained smears. Fixative solutions can prove problematic for downstream molecular extraction methods because of the potential to inhibit cellular lysis, because of the potential to interfere with the proteolytic enzymes used in this process, or due to cross-linking of nucleic acids and proteins (5, 6). Some prior studies have reported poor recovery of nucleic acids from SurePath and PreservCyt media and inferred that the DNA had degraded over time during storage (7-10). SurePath medium contains a low concentration of formalin, which is known to cross-link nucleic acids and protein (11,12). Formalin treatment results in reversible cross-linking of proteins to DNA, which can render it refractory to magnetic-or silica-based purification and subsequent downstream nucleic acid biochemistry, such as PCR (13). Cross-linking can be removed using proteinase K digestion and/or heat (14-16).We have developed a simple, one-step chemical lysis method for use with the BD hrHPV-GT assay that can process 0.5 ml of either LBC specimen without prior cell harvesting or the use of lytic enzymes. The method uses a combination of heat and chemicals to lyse LBC-preserved cells directly in the media. It also efficiently reverses cross-linking effects and allows biologically active DNA to be extracted directly from the resulting lysate (17-19).The objective of the current study was to examine the effect of long-term storage of LBC specimens at 2 ...

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“…For the majority of the isolates, gDNA was obtained directly by heat shock methods (24). A tip of the biomass pellet was picked using sterile toothpicks, placed in 50 l Tris-EDTA buffer, and subjected to heating at 95°C for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Phenotypic and Phylogenetic Identification of Coliform Bacteria Obtained Using 12 Coliform Methods Approved by the U.S. Environmental Protection Agency

Zhang

Hong

LeChevallier

et al. 2015

Appl Environ Microbiol

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The current definition of coliform bacteria is method dependent, and when different culture-based methods are used, discrepancies in results can occur and affect the accuracy of identification of true coliforms. This study used an alternative approach to the identification of true coliforms by combining the phenotypic traits of the coliform isolates and the phylogenetic affiliation of 16S rRNA gene sequences with the use of lacZ and uidA genes. A collection of 1,404 isolates detected by 12 U.S. Environmental Protection Agency-approved coliform-testing methods were characterized based on their phylogenetic affiliations and responses to their original isolation media and lauryl tryptose broth, m-Endo, and MI agar media. Isolates were phylogenetically classified into 32 true-coliform, or targeted Enterobacteriaceae (TE), groups and 14 noncoliform, or nontargeted Enterobacteriaceae (NTE), groups. It was shown statistically that detecting true-positive (TP) events is more challenging than detecting true-negative (TN) events. Furthermore, most false-negative (FN) events were associated with four TE groups (i.e., Serratia group I and the Providencia, Proteus, and Morganella groups) and most false-positive (FP) events with two NTE groups, the Aeromonas and Plesiomonas groups. In Escherichia coli testing, 18 out of 145 E. coli isolates identified by enzymatic methods were validated as FN. The reasons behind the FP and FN reactions could be explained through analysis of the lacZ and uidA genes. Overall, combining the analyses of the 16S rRNA, lacZ, and uidA genes with the growth responses of TE and NTE on culture-based media is an effective way to evaluate the performance of coliform detection methods. Total coliform bacteria and Escherichia coli are considered costeffective bacterial indicators for the protection of public health. Detectable total coliforms indicate potential contamination associated with the water distribution system, while E. coli is a good indicator of fecal contamination, with a health goal (i.e., maximum contaminant level goal [MCLG]) of zero under the Revised Total Coliform Rule (RTCR) (1). When a positive result of total-coliform testing is observed, public water systems (PWSs) are required to collect and analyze three repeat samples (1). A system assessment is required when a PWS exceeds a specific frequency of total-coliform occurrence. When an E. coli maximum contaminant level (MCL) violation incurs, a PWS must have an assessment performed by the state or a state-approved entity and must correct any sanitary defects (1). False-positive (FP) results for either total-coliform or E. coli tests impose an unnecessary burden on water utilities. On the other hand, false-negative (FN) results expose consumers to potential health threats and delay response times for effective action. Therefore, accurate detection of total coliforms and E. coli in drinking water systems is crucial for both water utilities and consumers.To date, the U.S. EPA has approved 12 methods for the detection of coliform bacteria i...

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Comparison of DNA extraction from cervical cells collected in PreservCyt solution for the amplification of Chlamydia trachomatis

Cited by 15 publications

References 39 publications

A feasibility study to identify proteins in the residual Pap test fluid of women with normal cytology by mass spectrometry-based proteomics

A feasibility study to identify proteins in the residual Pap test fluid of women with normal cytology by mass spectrometry-based proteomics

Long-Term Stability of Human Genomic and Human Papillomavirus DNA Stored in BD SurePath and Hologic PreservCyt Liquid-Based Cytology Media

Phenotypic and Phylogenetic Identification of Coliform Bacteria Obtained Using 12 Coliform Methods Approved by the U.S. Environmental Protection Agency

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