Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Amplicon sequencing”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 271 records · Page 15Linked to original sources
PubMed2005

The possibility of discriminating within the Bacillus cereus group using gyrB sequencing and PCR-RFLP.

Based on a combination of PCR and restriction endonuclease (RE) digestion (PCR-RE digestion), we have examined the possibility of differentiating members of the Bacillus cereus group. Fragments of the gyrB gene (362 bp) from pure cultures of 12 B. cereus, 25 B. thuringiensis, 25 B. mycoides and two B. anthracis strains were amplified and subsequently digested with Sau3A1. Furthermore, a majority of the amplicons were sequenced directly to verify the PCR-RE results. The results obtained suggest that only the B. mycoides generates specific fragments following PCR-RE. Conversely, it was not possible to discriminate between the B. cereus and the B. thuringiensis strains using the methods described.

Bacillus cereus↗
PubMed2002

PCR detection of psychrotolerant clostridia associated with deep tissue spoilage of vacuum-packed chilled meats.

AIMS: To develop a practical molecular procedure that directly (without isolation) and specifically detects the presence of clostridia, which cause the deep tissue spoilage condition. METHODS AND RESULTS: A primer set was designed and a PCR amplification procedure developed to detect the presence of Clostridium algidicarnis and Cl. putrefaciens 16S rDNA gene fragments in meat. The procedure yielded amplicons of the expected size with homologous DNA templates, but failed to give PCR products with DNAs from 47 food clostridia and common meat spoilage micro-organisms. The minimum level of detection was 10(4) cfu g-1 for nonenriched meat samples. Based on the established specificity of these primers, as well as DNA sequencing of amplicons, the presence of Cl. algidicarnis and/or Cl. putrefaciens was confirmed in a swab sample taken from the cartilage of an ovine stifle joint, which on opening exhibited strong offensive odours. CONCLUSIONS: The developed method can be used for rapid detection of clostridia causing deep tissue spoilage in commercial vacuum packs. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper reports practical procedures that can be used for rapid confirmation of the causative agents of deep tissue clostridial spoilage in commercial vacuum-packed chilled meats.

Animals↗
PubMed2001

[Identification of Chinese crude drug snake gallbladder by DNA molecular marker].

AIM: It is difficult to identify the Chinese crude drug snake gallbladder accurately by morphological and microscopical characteristics or chemical components only. In order to solve the problem, the technique based on DNA molecular marker was introduced into the authentication of snake gallbladder. METHODS: DNA templates were extracted from the membrane or the bile of snake gallbladder, and also from the muscle of the original animal Elaphe schrenckii. About 400 bp DNA fragments of 12S rRNA gene were amplified from the templates and sequenced subsequently. RESULTS: Enough amounts of DNA templates could be extracted from a bit of membrane or bile of snake gallbladder. The sequence of amplicons from the membrane, bile and muscle of the same individual were identical completely. CONCLUSION: The technique of DNA molecular marker could be used for the authentication of snake gallbladder and bile. The results indicate that the technique could be used for the identification of crude drugs from other animal secretion. DNA sequence analysis also demonstrated that the origins of commercial snake gallbladder were complicated and more efficient quality control was necessary for supervising the crude drug in the market.

Animals↗
PubMed2000

Screening the 3' region of the polycystic kidney disease 1 (PKD1) gene in 41 Bulgarian and Australian kindreds reveals a prevalence of protein truncating mutations.

Screening for disease-causing mutations in the unique region of the polycystic kidney disease 1 (PKD1) gene was performed in 41 unrelated individuals with autosomal dominant polycystic kidney disease. Exons 34-41 and 43-46 were assayed using PCR amplification and SSCP analysis followed by direct sequencing of amplicons presenting variant SSCP patterns. We have identified seven disease-causing mutations of which five are novel [c.10634-10656del; c.11587delG; IVS37-10C>A; c.11669-11674del; c.13069-13070ins39] and two have been reported previously [Q4010X; Q4041X]. Defects in this part of the gene thus account for 17% of our group of patients. Five of the seven sequence alterations detected are protein-truncating which is in agreement with mutation screening data for this part of the gene by other groups. The two other mutations are in-frame deletions or insertions which could destroy important functional properties of polycystin 1. These findings suggest that the first step toward cyst formation in PKD1 patients is the loss of one functional copy of polycystin 1, which indirectly supports the "two-hit" model of cystogenesis where a second somatic mutation inactivating the normal allele is necessary to occur for development of the disease condition.

3' Untranslated Regions↗
PubMed2001

PCR-SSCP comparison of 16S rDNA sequence diversity in soil DNA obtained using different isolation and purification methods.

This study compared different methods of direct DNA extraction and purification from a silt loam soil and investigated the relationship between DNA quantity and sequence diversity. Five extraction methods and four purification techniques were investigated. Quantities of DNA extracted were between 3.4+/-0.55 and 54.3+/-8.18 &mgr;g g(-1) (dry wt) of soil with OD(260)/OD(230) purity ratios between 0.80 and 1.15. Analysis of sequence diversity in all extracts was conducted using PCR-single strand conformation polymorphism (SSCP). Profiles generated using universal 16S rDNA primers (Com1/Com2) were found to be identical when used to amplify 16S rDNA extracted directly from soil. The genus Pseudomonas was targeted in order to reduce profile complexity, which was apparent when using universal 16S rDNA primers, and which hindered direct comparison of sequence diversity. A Pseudomonas culture library and non-cultured Pseudomonas 16S rDNA genes were used to provide a background count of Pseudomonas operational taxonomic units present in the soil. Cloning and sequencing of amplicons generated using a Pseudomonas-specific (Ps-for) and a universal 16S rDNA (Com2) primer, coupled with nested amplification (Com1/Com2 amplification from Ps-for/Ps-rev amplicons), used in conjunction with SSCP, revealed that environmental contaminants co-extracted with DNA, such as humic acid, significantly reduced primer specificity. SSCP was sensitive enough to reveal template bias in different primer sets. PCR-restriction fragment length-SSCP of Pseudomonas 16S rDNA amplified from soil-extracted DNA revealed distinct differences in sequence representation between extraction methods and showed that greater DNA yield is not synonymous with higher sequence diversity. We, therefore, suggest that DNA extractions from soil should be evaluated not only in terms of quantity and purity, but also in terms of the sequence diversity present. SSCP proved to be a valuable tool for the assessment of the methodologies commonly used in PCR-mediated microbial ecology studies.

Journal Article↗
PubMed1996

Shiga toxin-producing Escherichia coli strains from bovines: association of adhesion with carriage of eae and other genes.

Out of 174 bovine Shiga toxin-producing Escherichia coli (STEC) strains isolated from diarrheic calves in Germany and Belgium, 122 strains (70.1%) were selected because of their reactivity with the eae (E. coli attaching and effacing gene) probe ECW1-ECW2. One hundred seven of these eae-positive strains (87.7%) harbored stx1 genes, 13 strains (10.7%) had stx2 genes, and 2 strains (1.6%) had both stx genes. The strains displayed 17 different O types, the majority (97 strains) [79.5%]) belonging to O5 (5 strains), O26 (21 strains), O111 (13 strains) O118 (36 strains), O145 (9 strains), and O157 (13 strains). In the HEp-2 cell adhesion assay, 99 strains (81.1%) showed a localized adhesion, and 80 strains (65.6%) stimulated actin accumulation, as determined in the fluorescence actin staining test. None of the strains harbored genes coding for bundle-forming pili (bfpA), clearly differentiating them from enteropathogenic. E. cole. espB gene sequences were only detectable in 23 (18.9%) of the eae-positive bovine STEC strains. Three different PCRs were established, differentiating between eae sequences of enteropathogenic E. coli strain E2348/69 (O127:H6) and STEC strain EDL933 (O157: H7). Primers matching in the more heterologous downstream eae sequences gave amplicons in only 8 of the 17 O types (O84:H-, O103:H2, O111:H-, O111:H2, O119:H25, O128:H-, O145:H28, and O157:H-). Only 15 STEC strains, belonging to serotypes O111H:-, O111H:2, O145:H28, and O157:H-, gave amplicons in all three eae-specific PCRs. These data demonstrate that bovine STEC strains are a heterogeneous group of pathogenic bacteria, a lot of which share virulence markers with STEC strains causing infections in humans. However, in contrast to human STEC strains, bovine eae-positive STEC strains are mainly restricted to the stx1 genotype. The observation that espB sequences are not highly conserved might have consequences for the serological recognition of the ESPB protein in patients. Like in human STEC strains, eae-related sequences are closely associated with certain E. coli O groups; however, they are not serotype specific.

Actins↗
PubMed2005

Consensus sequence-based scheme for epidemiological typing of clinical and environmental isolates of Legionella pneumophila.

A previously described sequence-based epidemiological typing method for clinical and environmental isolates of Legionella pneumophila serogroup 1 was extended by the investigation of three additional gene targets and modification of one of the previous targets. Excellent typeability, reproducibility, and epidemiological concordance were determined for isolates belonging to both serogroup 1 and the other serogroups investigated. Gene fragments were amplified from genomic DNA, and PCR amplicons were sequenced by using forward and reverse primers. Consensus sequences are entered into an online database, which allows the assignment of individual allele numbers. The resulting sequence-based type or allelic profile comprises a string of the individual allele numbers separated by commas, e.g., 1,4,3,1,1,1, in a predetermined order, i.e., flaA, pilE, asd, mip, mompS, and proA. The index of discrimination (D) obtained with these six loci was calculated following analysis of a panel of 79 unrelated clinical isolates. A D value of > 0.94 was obtained, and this value appears to be sufficient for use in the epidemiological investigation of outbreaks caused by L. pneumophila. The D value rose to 0.98 when the results of the analysis were combined with those of monoclonal antibody subgrouping. Sequence-based typing of L. pneumophila is epidemiologically concordant and discriminatory, and the data are easily transportable. This consensus method will assist in the epidemiological investigation of L. pneumophila infections, especially travel-associated cases, by which it will allow a rapid comparison of isolates obtained in more than one country.

Bacterial Typing Techniques↗
PubMed2003

PCR detection of psychrophilic Clostridium spp. causing 'blown pack' spoilage of vacuum-packed chilled meats.

AIMS: To develop a practical molecular procedure that directly, without isolation, and specifically detects the presence of clostridia which cause 'blown pack' spoilage of vacuum-packed meat. METHODS AND RESULTS: Primer sets and PCR amplification procedures were developed that detect the presence of 16S rDNA gene and/or 16S-23S rDNA internal transcribed spacer fragments of 'blown pack' causing clostridia in meat. The specificity of the developed procedures was evaluated with DNA obtained from close phylogenetic neighbours of 'blown pack' causing clostridia, food clostridia and common meat spoilage microorganisms. The sensitivity of detection was assessed in non-enriched and low-temperature-enriched beef mince inoculated with serially diluted pure cultures of Clostridium estertheticum DSMZ 8809T and Cl. gasigenes DB1AT. The efficacy of detection procedures was evaluated for naturally contaminated vacuum-packed meat samples. Three primer sets, 16SE, 16SDB and EISR, produced amplicons of the expected size with DNA templates from target clostridia, but failed to yield PCR products with DNAs from any other microorganisms tested. With 16SE and 16SDB primers, minimum levels of detection were 104 CFU g(-1) for non-enriched, and 102 CFU g(-1) for enriched meat samples. Based on the established specificity of these primers, as well as DNA sequencing of amplicons, Cl. gasigenes was confirmed as the causative agent of 'blown pack' spoilage in two packs, and Cl. estertheticum as the causative agent in the third. CONCLUSIONS: The developed method can be used for rapid detection of 'blown pack' causing clostridia in commercial blown packs, or following low temperature enrichment, for detection of these microorganisms in meat containing as few as 100 clostridial cells per gram. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper reports practical procedures that can be used for rapid confirmation of the causative agents of clostridial 'blown pack' spoilage in commercial spoiled packs, or for detection of psychrophilic clostridia in epidemiological trace back of 'blown pack' spoilage incidents in meat processing plants.

Clostridium↗
PubMed1998

Mutations in NS5A region of hepatitis C virus genome correlate with presence of NS5A antibodies and response to interferon therapy for most common European hepatitis C virus genotypes.

A part of the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) amino acid sequence, designated as an interferon (IFN)-sensitive determining region (ISDR), has been shown to be correlated with a response to IFN in Japanese patients. We have shown previously that the presence of NS5A antibodies (Abs) detected by the INNOLIA test (IL-NS5A Ab) is also correlated with a response to IFN. The aim of this study was to investigate, in a wide range of patients, the possible relationship within the NS5A protein between the sequence of ISDR and that used in the INNOLIA test designated as IL3R. Serum samples from 52 patients infected by HCV genotypes 1, 2, and 3 were analyzed before and after treatment. The patients were classified as nonresponders (NRs), responder-relapsers (RRs), or long-term responders (LTRs). We amplified the NS5A region for 42 patients using polymerase chain reaction (PCR), and these amplicons were sequenced directly. The 10 remaining patients were analyzed using PCR with mutation-specific primers. No correlation was found between the IL3R sequence of the HCV strains and the presence of the IL-NS5A Ab for all genotypes. However, for the subtype 1b, only 2 of 11 NR patients tested had an arginin in position 2218 within the ISDR versus 3 of 3 LTR and 10 of 13 RR patients. All patients with R-2218 had IL-NS5A Ab. For the genotype 1a, 2 of 2 LTR and 1 of 3 RR were mutated in position 2216-2218 in comparison to three NR sequences. For the genotype 3, no mutations were found in the region homologous to 1b-ISDR, but 4 of 5 LTR and RR patients had a mutation T-2161 to A or V versus 0 of 3 NR patients. A close correlation was found between arginin in position 2218 in ISDR, the presence of IL-NS5A Ab, and the response to IFN therapy for genotype 1b, but this association did not predict a long-term response. For genotype 3, a potential ISD mutation could be located at the codon 2161.

Amino Acid Sequence↗
PubMed1995

Trypanosoma cruzi: parasite detection and strain discrimination in chronic chagasic patients from northeastern Brazil using PCR amplification of kinetoplast DNA and nonradioactive hybridization.

Blood samples from 172 individuals from northeastern Brazil were subjected to PCR amplification of Trypanosoma cruzi-specific kDNA sequences. This method enabled us to detect parasite DNA in 21 of 47 patients that were serologically positive. In addition, 1 patient that gave doubtful results with chagasic serology was confirmed as positive by PCR. We applied the same PCR detection method to the feces of wild triatomines captured in the same region, obtaining three positive results that were confirmed by microscopic examination. The 25 amplified products obtained in this study were then reamplified with primers that gave a final amplicon containing sequences from the most variable region of kDNA minicircles. These were used as probes in hybridization experiments aimed at defining the degree of relatedness between the strains infecting humans and insects based on kDNA homologies. We found that the amplification products from the three triatomines were related and showed no cross-hybridization with those obtained from human infections. Eight amplified products from human infections showed no cross-hybridization and did not hybridize with products from other patients. This indicates that the strains of T. cruzi circulating in the region present a high level of genetic heterogeneity. Finally, a number of amplified products hybridized with amplicons that did not hybridize with each other, indicating that infections with a parasite population presenting a mixed kDNA content (either due to different strains of T. cruzi or to a hybrid parasite) are a more frequent event than previously thought.

Animals↗
PubMed2004

Comprehensive detection and identification of human coronaviruses, including the SARS-associated coronavirus, with a single RT-PCR assay.

The SARS-associated human coronavirus (SARS-HCoV) is a newly described, emerging virus conclusively established as the etiologic agent of the severe acute respiratory syndrome (SARS). This study presents a single-tube RT-PCR assay that can detect with high analytical sensitivity the SARS-HCoV, as well as several other coronaviruses including other known human respiratory coronaviruses (HCoV-OC43 and HCoV-229E). Species identification is provided by sequencing the amplicon, although a rapid screening test by restriction enzyme analysis has proved to be very useful for the analysis of samples obtained during the SARS outbreak in Toronto, Canada.

Base Sequence↗
PubMed2000

The heteroduplex mobility assay (HMA) as a pre-sequencing screen for Norwalk-like viruses.

Molecular epidemiological studies of Norwalk-like viruses (NLVs), previously known as small round structured viruses (SRSVs), are dependant currently on DNA sequencing of PCR amplicons, which is expensive and time consuming. The Heteroduplex Mobility assay (HMA) was evaluated as a method for identification of PCR amplicons from the commonly circulating NLV strains without DNA sequencing. The procedure was developed for use with two reference strains, a Mexico virus-like strain (MXV-like; Hu¿NLV¿RBH¿1993¿UK) and the Grimsby virus strain (Hu¿NLV¿Gimsby¿1995¿UK), and was optimised with regards to the annealing and electrophoresis conditions and the electrophoresis gel matrix. Using the optimised conditions, amplicons of less than 90% sequence identity formed visible heteroduplexes, allowing the strains to be placed into three categories; Mexico-like, Grimsby-like and non-Mexico virus/non-Grimsby virus strains. Outbreak strains 'genotyped' previously by DNA sequencing as Mexico virus or Grimsby virus were identified correctly by the heteroduplex mobility assay. The procedure was applied prospectively to strains from 130 outbreaks occurring in the UK between 1997 and 1998. Heteroduplex mobility assay was successful on 120 (92%) strains of which 68 (57%) were GRV-like strains, three (2.5%) were Mexico virus-like strains and 49 (41%) were categorised as non- Mexico/non-Grimsby virus strains. Amplicons from 50 of the 120 strains were sequenced and there was perfect correlation between the heteroduplex mobility assay categorisation and phylogenetic analysis. HMA offers a rapid, robust and far cheaper alternative to sequencing for the identification of prevalent Norwalk-like virus genotypes for molecular epidemiological studies.

Caliciviridae Infections↗
PubMed1999

Hepatitis cRNA quasispecies complexity in patients with alcoholic liver disease.

Alcohol abuse is described as a major cofactor in the development of hepatitis C (HCV) associated liver disease and may play a role in the outcome of interferon-based treatment interventions. The association between HCV viral heterogeneity and alcohol has not been previously described. This study was designed to evaluate the quasispecies nature of the HCV population in patients with compensated and decompensated alcoholic liver disease, to test the hypothesis that alcoholics have greater complexity than matched nonalcoholic controls. A nonisotopic heteroduplex complexity assay (HCA) was first validated by comparison with results of quasispecies complexity determined by subcloning and sequencing of amplicon products from the E2/NS1 hypervariable coding region (HVR). Subsequently, this methodology was applied to comparison of 20 compensated (Child's-Pugh A) and decompensated (Child's-Pugh B/C) alcoholic and 20 nonalcoholic controls. The complexity of the HVR, as well as the 5' Untranslated (5'UT) and the NS5b coding domains were evaluated. The HCA methodology provided a reasonable semiquantitative measure of HCV RNA quasispecies variability compared with subclone sequence homology comparison. Overall, alcoholic patients had greater quasispecies complexity (2.65 bands) than nonalcoholic controls (1.6 bands; P =.01). Subset analysis revealed that compensated alcoholic patients had a mean of 3.1 homo/heteroduplex bands per sample whereas Child's-Pugh B/C alcoholics showed intermediate complexity. A similar quasispecies complexity difference was seen in the 5'UTR, but not in the NS5b coding domain. Quasispecies complexity was not associated with viral titer, complementary DNA concentration, or genotype. The differences in quasispecies complexity may help explain reports of poor interferon responsiveness in alcoholic patients.

5' Untranslated Regions↗
PubMed2026

Optimization of a Semi-nested PCR Protocol for Amplifying the Entire Spike Protein Region to Identify SARS-CoV-2 Variants in Wastewater.

Wastewater-based monitoring of SARS-CoV-2 and other pathogens is a widely adopted tool for assessing epidemic dynamics. While quantitative assays are commonly used to estimate infection levels in catchment populations, phylogenetic information-such as identifying circulating variants-is also crucial for public health. However, applying the widely used ARTIC protocol for full-genome sequencing to wastewater samples has proven challenging, likely due to the limited specificity and sensitivity of multiplex RT-PCR in such complex matrices. In this study, we developed and optimized a semi-nested RT-PCR assay targeting the full S-protein coding region (~4000 bases) for phylogenetic characterization of SARS-CoV-2 in wastewater. By reducing multiplexing and using single-plex reactions for both RT and PCR steps, we successfully amplified ~2000&#xa0;bp fragments. Amplicons were sequenced using the Flongle Flow Cell platform. The optimized method-consisting of reverse transcription with specific primers followed by three parallel single-plex semi-nested PCRs-yielded over 1,000 SARS-CoV-2-like reads per primer set in 30 out of 39 wastewater samples in treatment plants in Japan, including those with <10 copies per analyte. Variant proportions were estimated using a newly developed approach based on single-nucleotide variant pattern matrix, revealing the presence of multiple co-circulating variants, including XBB lineages, JN.1, and notably BA.2.75, which was undetected in domestic clinical surveillance. These results highlight the effectiveness of our approach for detecting temporal shifts in SARS-CoV-2 variants, even at low RNA concentrations.

Polymerase Chain Reaction↗
PubMed1999

Somatic mutation analysis of IgH variable regions reveals that tumor cells of most parafollicular (monocytoid) B-cell lymphoma, splenic marginal zone B-cell lymphoma, and some hairy cell leukemia are composed of memory B lymphocytes.

The cell of origin of parafollicular (monocytoid) B cell lymphoma (PBCL), splenic marginal zone lymphoma (SMZL), and hairy cell leukemia (HCL) is controversial. To better understand the relationship between these low-grade B-cell neoplasms, we analyzed the nucleotide sequences of the rearranged immunoglobulin heavy (IgH) chain variable (V) region of the clonal population of cells in five cases of PBCL, four cases of SMZL, and seven cases of HCL to determine whether these neoplasms could be differentiated by the degree of somatic mutation in the IgH V gene or by the IgH V gene family usage. DNA was extracted from diagnostic material and clonality confirmed by PCR. The DNA was reamplified using V heavy chain family specific primers, and the amplicons were sequenced. Sequences were compared with germline IgH V gene sequences, and base changes were determined to be silent or to represent amino acid replacements by using three different methods. Four of five (80%) cases of PBCL, three of four (75%) cases of SMZL, and three of seven (43%) cases of HCL showed evidence of antigen selection, suggesting that these neoplasms involved clonal expansions of postgerminal center memory lymphocytes. Only SMZL showed a preferential usage of V(H)1 family genes.

Adult↗
PubMed2002

Long PCR-based amplification of the entire mitochondrial genome from single parasitic nematodes.

Mitochondrial genome sequences provide useful markers for investigating population genetic structures because of their maternal inheritance and high evolutionary rates. There is, however, a paucity of information on mitochondrial genomes for many parasitic organisms, including nematodes, which appears to relate mainly to technical limitations and (for modestly funded laboratories) the cost associated with full mitochondrial genome sequencing. In this article, we describe a simple, relatively inexpensive long-PCR approach for the amplification (using two sets of primers) of the entire mitochondrial genome from individual parasitic nematodes for subsequent sequencing, which overcomes these limitations. We employed two species of human hookworm (Ancylostoma duodenale and Necator americanus; order Strongylida) to establish the long-PCR conditions, and then extended its use to a number of other species of parasitic nematode of the class Secernentea (orders Strongylida, Ascaridida and Rhabditida). The long-PCR method for the amplification of the entire mitochondrial genome from single nematodes, coupled with direct sequencing of amplicons, provides a useful tool for the comparative analysis of genome organisation and evolution of a range of nematode groups. It also creates a platform for molecular ecological and population genetic studies.

Ancylostoma↗
PubMed1999

Development of a PCR assay for rapid detection of enterococci.

Enterococci are becoming major nosocomial pathogens, and increasing resistance to vancomycin has been well documented. Conventional identification methods, which are based on culturing, require 2 to 3 days to provide results. PCR has provided a means for the culture-independent detection of enterococci in a variety of clinical specimens and is capable of yielding results in just a few hours. However, all PCR-based assays developed so far are species specific only for clinically important enterococci. We have developed a PCR-based assay which allows the detection of enterococci at the genus level by targeting the tuf gene, which encodes elongation factor EF-Tu. Initially, we compared the nucleotide sequences of the tuf gene from several bacterial species (available in public databases) and designed degenerate PCR primers derived from conserved regions. These primers were used to amplify a target region of 803 bp from four enterococcal species (Enterococcus avium, E. faecalis, E. faecium, and E. gallinarum). Subsequently, the complete nucleotide sequences of these amplicons were determined. The analysis of a multiple alignment of these sequences revealed regions conserved among enterococci but distinct from those of other bacteria. PCR primers complementary to these regions allowed amplification of genomic DNAs from 14 of 15 species of enterococci tested (E. solitarius DNA could not be amplified). There was no amplification with a majority of 79 nonenterococcal bacterial species, except for 2 Abiotrophia species and several Listeria species. Furthermore, this assay efficiently amplified all 159 clinical isolates of enterococci tested (61 E. faecium, 77 E. faecalis, 9 E. gallinarum, and 12 E. casseliflavus isolates). Interestingly, the preliminary sequence comparison of the amplicons for four enterococcal species demonstrated that there were some sequence variations which may be used to generate species-specific internal probes. In conclusion, this rapid PCR-based assay is capable of detecting all clinically important enterococci and has potential for use in clinical microbiology laboratories.

Bacterial Typing Techniques↗
PubMed2003

PCR-restriction fragment length polymorphism method for detection of Cyclospora cayetanensis in environmental waters without microscopic confirmation.

We developed an alternative nested-PCR-restriction fragment length polymorphism (RFLP) protocol for the detection of Cyclospora cayetanensis in environmental samples that obviates the need for microscopic examination. The RFLP method, with the restriction enzyme AluI, differentiates the amplified target sequence from C. cayetanensis from those that may cross-react. This new protocol was used to reexamine a subset (121 of 180) of surface water samples. Samples previously positive when the CYCF3E and CYCR4B primers (33) and RFLP with MnlI (20) were used were also PCR positive with the new primers; however, they were RFLP negative. We verified, by sequencing these amplicons, that while two were most likely other Cyclospora species, they were not C. cayetanensis. We can detect as few as one oocyst seeded into an autoclaved pellet flocculated from 10 liters of surface water. This new protocol should be of great use for environmental microbiologists and public health laboratories.

Base Sequence↗