Privacy Risks from Genomic Data-Sharing Beacons

The human genetics community needs robust protocols that enable secure sharing of genomic data from participants in genetic research. Beacons are web servers that answer allele-presence queries—such as “Do you have a genome that has a specific nucleotide (e.g., A) at a specific genomic position (e.g., position 11,272 on chromosome 1)?”—with either “yes” or “no.” Here, we show that individuals in a beacon are susceptible to re-identification even if the only data shared include presence or absence information about alleles in a beacon. Specifically, we propose a likelihood-ratio test of whether a given individual is present in a given genetic beacon. Our test is not dependent on allele frequencies and is the most powerful test for a specified false-positive rate. Through simulations, we showed that in a beacon with 1,000 individuals, re-identification is possible with just 5,000 queries. Relatives can also be identified in the beacon. Re-identification is possible even in the presence of sequencing errors and variant-calling differences. In a beacon constructed with 65 European individuals from the 1000 Genomes Project, we demonstrated that it is possible to detect membership in the beacon with just 250 SNPs. With just 1,000 SNP queries, we were able to detect the presence of an individual genome from the Personal Genome Project in an existing beacon. Our results show that beacons can disclose membership and implied phenotypic information about participants and do not protect privacy a priori. We discuss risk mitigation through policies and standards such as not allowing anonymous pings of genetic beacons and requiring minimum beacon sizes.     

Target discrimination by surface-immobilized molecular beacons designed to detect Francisella tularensis

A molecular beacon (MB) array was designed based on unique regions of the 16S rRNA of the bacterium Francisella tularensis. Nucleic acid molecular beacons undergo a spontaneous fluorogenic conformational change when they hybridize to specific complementary targets. The array was printed on aldehyde glass or hydrogel slides and evaluated for functioning in presence of complementary oligonucleotide sequences, single-nucleotide mismatch sequences and multiple nucleotide mismatch sequences. Discriminating true target from mismatched targets was found to be dependent on type, number, and location of mismatches within the beacon (i.e. located in the stem or loop regions). Optimal conditions for molecular beacon deposition, and target hybridization were determined for oligonucleotide target mismatch discrimination. The beacon array was stable upon recharging by exposure to an alkaline solution, and repeatedly used. In addition, performance of the beacon array biosensor was compared with molecular beacons in homogeneous solution.     

Extraction of ribosomal RNA from soil for detection of Frankia with oligonucleotide probes

Sequences of 16S rRNA of the nitrogen-fixing Frankia strain Ag45/Mut15 and the ineffective Frankia strain AgB1.9 were used to design a genus-specific oligonucleotide probe. Hybridization experiments of this Frankia probe and a second probe, specific for Nif⁺-Frankia strains only, were used to detect Frankia specific target sequences in RNA isolations from soil. A method is described for direct isolation of RNA from a loamy soil and a peat. Yields of about 10 ng RNA/g wet soil are obtained without detectable contamination with humic acids. Isolation of RNA after initial extraction of bacteria from soil resulted in significantly lower RNA yields, compared to the direct isolation procedure. Hybridization with both probes against rRNA isolations from Frankia-containing soil could detect target sequences within RNA isolations from 1 g wet soil with an estimated detection limit of 10⁴ cells.     

Improving the performance of an end-point PCR assay commonly used for the detection of Bacteroidales pertaining to cow feces

Bacteroidales are normal gut flora of warm-blooded animals. Since each host species carries a different diversity of Bacteroidales, the detection of host-associated gene markers of Bacteroidales has emerged as a promising tool for the tracking of the source of fecal pollution in aquatic ecosystems. To detect cow-associated Bacteroidales, a commonly used method has been an end-point PCR assay with the 16S rRNA genes primers CF128F (cow-associated) and Bac708R (all Bacteroidales). The PCR assay has demonstrated high rates of true-positive detection (i.e., high sensitivity) in all previous studies. However, the assay also had high rates of false-positive detection to the samples of non-target hosts in some cases (i.e., low specificity). In opposite to the reason many investigators have proposed, our results suggested that false detection was not necessarily due to the presence of the target sequence of CF128F in the feces of non-target hosts. Instead, we found sequences of non-target hosts having single internal mismatches with CF128F. Those mismatches were well tolerated in PCR, partly due to the universality of Bac708R. To improve the detection performance, we designed a novel primer CF592R (targeting the same clade of sequences as CF128F) to substitute Bac708R. The use of CF529R alleviated false detection and also led to a tenfold reduction in detection limit in the samples tested, compared to the use of Bac708R. Many other end-point PCR assays that detect the 16S rRNA genes in Bacteroidales also use a host-associated primer to couple with Bac708R, and low specificity or sensitivity has been reported. Based on our findings for CF128F, we suggest that the suitability of Bac708R in those PCR assays needs to be revisited.     

Molecular Method for Detection of Total Coliforms in Drinking Water Samples

This work demonstrates the ability of a bacterial concentration and recovery procedure combined with three different PCR assays targeting the lacZ, wecG, and 16S rRNA genes, respectively, to detect the presence of total coliforms in 100-ml samples of potable water (presence/absence test). PCR assays were first compared to the culture-based Colilert and MI agar methods to determine their ability to detect 147 coliform strains representing 76 species of Enterobacteriaceae encountered in fecal and environmental settings. Results showed that 86 (58.5%) and 109 (74.1%) strains yielded a positive signal with Colilert and MI agar methods, respectively, whereas the lacZ, wecG, and 16S rRNA PCR assays detected 133 (90.5%), 111 (75.5%), and 146 (99.3%) of the 147 total coliform strains tested. These assays were then assessed by testing 122 well water samples collected in the Québec City region of Canada. Results showed that 97 (79.5%) of the samples tested by culture-based methods and 95 (77.9%), 82 (67.2%), and 98 (80.3%) of samples tested using PCR-based methods contained total coliforms, respectively. Consequently, despite the high genetic variability of the total coliform group, this study demonstrated that it is possible to use molecular assays to detect total coliforms in potable water: the 16S rRNA molecular assay was shown to be as efficient as recommended culture-based methods. This assay might be used in combination with an Escherichia coli molecular assay to assess drinking water quality.     

Specific detection of an oil-degrading bacterium, Corynebacterium sp. IC10, in sand microcosms by PCR using species-specific primers based on 16S rRNA gene sequences

A species-specific PCR technique to detect an oil-degrading bacterium, Corynebacterium sp. IC10, released into sand microcosms is described. PCR primers, specific to strain IC10, were designed based on 16S rRNA gene sequences and tested against both closely and distantly related bacterial strains using four primer combinations involving two forward and two reverse primers. Two sets of them were specific to the strain IC10 and Corynebacterium variabilis and one set was selected for further analysis. The PCR amplification was able to detect 1 pg template DNA of strain IC10 and 1.2×10⁴ c.f.u. of IC10 ml wet sand^–1 in the presence of 3×10⁸ Escherichia coli cells. In non-sterile sand microcosms seeded with the strain IC10, the sensitivity of detection decreased to 9.6×10⁵ c.f.u. ml wet sand^–1. The detection sensitivity thus depends on the complexity of background heterogeneous DNA of environmental samples. The assay is suitable for detection of Corynebacterium sp. IC10 in laboratory microcosms, however, cross reaction with non-oil degrading coryneforms may prohibit its use in uncharacterized systems.     

An E. coli 5S rRNA Deletion Mutant Useful for the Study of 5S rRNA Structure/Function Relationships

We report on the construction of a novel strain of E. coli that can be useful for studies on the structure/function relationship of 5S rRNAs. The bacterial strain is deficient in six of the eight naturally occurring 5S rRNA genes (operons B, D, H, G, E) and demonstrates a greatly reduced growth rate that can be compensated by the plasmid-encoded expression of 5S rRNA. The relatively large difference in growth rate between compensated and non-compensated mutants provides the basis for a quick and simple assaying system for both the evaluation and mass screening of divergent 5S rRNA sequences for function. We describe the construction of the 5S rRNA deletion mutant BDHGE and characterize the usefulness and limitations of the system for evaluating structure/function relationships of 5S rRNA sequence. Received: 20 August 2000 / Accepted: 2 January 2001     

Development of a multiplex and real time PCR assay for the specific detection of Arcobacter butzleri and Arcobacter cryaerophilus

A new multiplex PCR and two specific TaqMan assays were developed to target the emerging pathogens A. butzleri and A. cryaerophilus. The assays also included an internal control to verify the presence of bacterial target DNA and amplification integrity. The multiplex assay used a published primer set (CRY1 and CRY2) for detecting A. cryaerophilus DNA (Houf, K., Tutenel, A., De Zutter, L., Van Hoof, J. and Vandamme, P., 2000. Development of a multiplex PCR assay for the simultaneous detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii. FEMS microbiology letters, 193 (1): 89-94.) and a novel A. butzleri primer set designed to target the rpoB/C gene sequences. To improve sample throughput and assay sensitivity a TaqMan assay for each Arcobacter spp. was developed which again utilised the heterogeneity contained in the rpoB/C and 23s rRNA gene sequences. The two TaqMan assays provided >2 log improvement in detection sensitivity for both Arcobacter spp. compared with the multiplex PCR assay and were able to detect <10 CFU per PCR reaction. To evaluate the effectiveness of the Arcobacter TaqMan assays with field isolates the assays were used to screen DNA samples prepared from faecal, hide and environmental samples obtained from two meat processing plants. In these studies, the TaqMan assays revealed that 2/150 (1.3%) samples were A. butzleri-positive, 11/150 (7.3%) were A. cryaerophilus-positive and the identity of generated amplicons was confirmed by DNA sequencing. Our results show that these TaqMan assays provide improvements in sensitivity and species-representation over other published Arcobacter PCR assays and they are compatible with detecting Arcobacters in sub-optimal matrices.     

Molecular Identification and Typing of Putative Probiotic Indigenous Lactobacillus plantarum Strain Lp91 of Human Origin by Specific Primed-PCR Assays

In the present scenario, it is now well documented that probiotics confer health benefits to the host and the purported probiotic effects are highly strain specific. Hence, accurate genotypic identification is extremely important to link the strain to the specific health effect. With this aim, specific primed-PCR assays were developed and explored for the molecular identification and typing of a putative indigenous probiotic isolate Lp91 of human faecal origin. PCR with specific primers targeting 23S rRNA gene of genus Lactobacillus and 16S rRNA gene of species L. plantarum resulted positive for Lp91. In addition, BLAST analysis of 16S rRNA gene sequence of Lp91 and multiple sequence alignment of 16S rRNA gene variable (V2-V3) regions along with the reference sequences revealed it as L. plantarum with a sequence identity of more than 99%. Furthermore, resolution of 16S rRNA gene sequences was sufficient to infer a phylogenetic relationship amongst Lactobacillus species. In order to determine strain-level variations, randomly amplified polymorphic DNA (RAPD) banding profiles of Lp91 obtained with OPAA-01, OPAP-01 and OPBB-01 primers were compared with those of reference strains of Lactobacillus spp., and Lp91 could be delineated as a distinct strain. Apart from this, presence of probiotic markers viz. bile salt hydrolase (bsh) and collagen-binding protein (cbp) encoding genes in Lp91 genome could be attributed to its exploitation as a potential probiotic adjunct in the development of indigenous functional foods. Lactobacillus isolates/or strains from the gastrointestinal system, fermented products and other environmental niches could be identified and characterized by employing the PCR methods developed in this study; they are rapid, reproducible and more accurate than the conventional methods based on the fermentation profiles.     

Direct‐geneFISH: a simplified protocol for the simultaneous detection and quantification of genes and rRNA in microorganisms

Although fluorescence in situ hybridization (FISH) with specific ribosomal RNA (rRNA)‐targeted oligonucleotides is a standard method to detect and identify microorganisms, the specific detection of genes in bacteria and archaea, for example by using geneFISH, requires complicated and lengthy (> 30 h) procedures. Here we report a much improved protocol, direct‐geneFISH, which allows specific gene and rRNA detection within less than 6 h. For direct‐geneFISH, catalyzed amplification reporter deposition (CARD) steps are removed and fluorochrome‐labelled polynucleotide gene probes and rRNA‐targeted oligonucleotide probes are hybridized simultaneously. The protocol allows quantification of gene copy numbers per cell and the signal of the directly labelled probes enables a subcellular localization of the rRNA and target gene. The detection efficiencies of direct‐geneFISH were first evaluated on Escherichia coli carrying the target gene on a copy‐control vector. We could show that gene copy numbers correlated to the geneFISH signal within the cells. The new protocol was then applied for the detection of the sulfate thiolhydrolase (soxB) genes in cells of the gammaproteobacterial clade SUP05 in Lake Rogoznica, Croatia. Cell and gene detection efficiencies by direct‐geneFISH were statistically identical to those obtained with the original geneFISH, demonstrating the suitability of the simpler and faster protocol for environmental samples.     

Use of DNA and Peptide Nucleic Acid Molecular Beacons for Detection and Quantification of rRNA in Solution and in Whole Cells

DNA and peptide nucleic acid (PNA) molecular beacons were successfully used to detect rRNA in solution. In addition, PNA molecular beacon hybridizations were found to be useful for the quantification of rRNA: hybridization signals increased in a linear fashion with the 16S rRNA concentrations used in this experiment (between 0.39 and 25 nM) in the presence of 50 nM PNA MB. DNA and PNA molecular beacons were successfully used to detect whole cells in fluorescence in situ hybridization (FISH) experiments without a wash step. The FISH results with the PNA molecular beacons were superior to those with the DNA molecular beacons: the hybridization kinetics were much faster, the signal-to-noise ratio was much higher, and the specificity was much better for the PNA molecular beacons. Finally, it was demonstrated that the combination of the use of PNA molecular beacons in FISH and flow cytometry makes it possible to rapidly collect quantitative FISH data. Thus, PNA molecular beacons might provide a solution for limitations of traditional FISH methods, such as variable target site accessibility, poor sensitivity for target cells with low rRNA content, background fluorescence, and applications of FISH in microfluidic devices.     

Use of DNA and peptide nucleic acid molecular beacons for detection and quantification of rRNA in solution and in whole cells

DNA and peptide nucleic acid (PNA) molecular beacons were successfully used to detect rRNA in solution. In addition, PNA molecular beacon hybridizations were found to be useful for the quantification of rRNA: hybridization signals increased in a linear fashion with the 16S rRNA concentrations used in this experiment (between 0.39 and 25 nM) in the presence of 50 nM PNA MB. DNA and PNA molecular beacons were successfully used to detect whole cells in fluorescence in situ hybridization (FISH) experiments without a wash step. The FISH results with the PNA molecular beacons were superior to those with the DNA molecular beacons: the hybridization kinetics were much faster, the signal-to-noise ratio was much higher, and the specificity was much better for the PNA molecular beacons. Finally, it was demonstrated that the combination of the use of PNA molecular beacons in FISH and flow cytometry makes it possible to rapidly collect quantitative FISH data. Thus, PNA molecular beacons might provide a solution for limitations of traditional FISH methods, such as variable target site accessibility, poor sensitivity for target cells with low rRNA content, background fluorescence, and applications of FISH in microfluidic devices.     

Design of a core–shell type immuno-magnetic separation system and multiplex PCR for rapid detection of pathogens from food samples

We report an immuno-magnetic separation system developed by the immobilization of pathogen-specific antibodies on the core–shell magnetic beads. The magnetic beads were grafted with glycidylmethacrylate (GMA) using surface-initiated atom transfer radical polymerization (SI-ATRP). For immuno-magnetic separation (IMS) of target bacterial cells from others, antibodies for Escherichia coli and Salmonella enterica serovar Typhimurium cells were immobilized on the magnetic beads via glutaraldehyde coupling reaction. Our IMS system successfully separated Salmonella cells when the concentrations of target (i.e., Salmonella) and interfering (i.e., E. coli) cells were at the same level. Polymerase chain reaction (PCR) assays amplifying the rfb/rfbE region of the E. coli genome and a 647-bp fragment of the invA region of Salmonella were performed as the specific selection to accurately confirm the presence of E. coli and Salmonella, respectively. IMS and multiplex PCR methods can be used for specific and quantitative detection of pathogens from food samples. Thus, this study developed a reliable and direct system for rapid detection of Salmonella and E. coli in food samples. In addition, IMS method could be easily adapted to detect other pathogens by selecting the pertinent antibody.     

First detection of “Candidatus Phytoplasma asteris” - and “Candidatus Phytoplasma solani”-related strains in fig trees

In July 2017, a survey was conducted in a fig collection plot at Locorotondo (south of Italy) to investigate the possible presence of phytoplasmas in plants showing yellowing, deformed leaves, short internodes, mottling and mosaic. Samples were collected from symptomatic plants and tested by nested PCR assays using universal and specific primers to amplify the 16S rDNA of these prokaryotes. PCR results detected the presence of phytoplasma sequences in twenty plant samples that resulted clustering two phylogenetically distinct phytoplasmas, i.e., “Candidatus Phytoplasma asteris” and “Candidatus Phytoplasma solani” affiliated to 16SrI and 16SrXII ribosomal groups, respectively. The presence of phytoplasmas belonging to both ribosomal groups was confirmed with group specific quantitative PCR and RFLP assays on 16S ribosomal amplicons. Results of this study indicate for the first time the occurrence of phytoplasmas in fig; however, more work should be carried out to verify their association with the symptoms observed on diseased fig plants.     

Direct detection of genomic DNA with fluidic force discrimination assays

Herein, we describe the direct detection of genomic DNA using fluidic force discrimination (FFD) assays. Starting with extracted bacterial DNA, samples are fragmented by restriction enzymes or sonication, then thermocycled in the presence of blocking and labeling sequences, and finally detected with microbead-based FFD assays. Both strain and species identification of extracted Bacillus DNA have been demonstrated in <30 min, without amplification (e.g., PCR). Femtomolar assays can be achieved with this rapid and simple procedure.     

Development of PCR Assays Targeting Genes in O-Antigen Gene Clusters for Detection and Identification of Escherichia coli O45 and O55 Serogroups

The Escherichia coli O45 O-antigen gene cluster of strain O45:H2 96-3285 was sequenced, and conventional (singleplex), multiplex, and real-time PCR assays were designed to amplify regions in the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes. In addition, PCR assays targeting the E. coli O55 wzx and wzy genes were designed based on previously published sequences. PCR assays targeting E. coli O45 showed 100% specificity for this serogroup, whereas by PCR assays specific for E. coli O55, 97/102 strains serotyped as E. coli O55 were positive for wzx and 98/102 for wzy. Multiplex PCR assays targeting the E. coli O45 and the E. coli O55 wzx and wzy genes were used to detect the organisms in fecal samples spiked at levels of 10⁶ and 10⁸ CFU/0.2 g feces. Thus, the PCR assays can be used to detect and identify E. coli serogroups O45 and O55.     

Anti-DNA·RNA antibodies: an efficient tool for non-isotopic detection of Listeria species through a liquid-phase hybridization assay

This study was undertaken to evaluate the potential of a new approach using anti-DNA · RNA monoclonal antibodies to detect Listeria in both pure culture and inoculated meat and meat products. A sensitive liquid-phase assay was first developed, based on the formation in solution of a hybrid between a 784-bp DNA probe, specific for the genus Listeria, and target rRNA. Monoclonal antibody and antisera raised against hybrid nucleic acids were then used in various immunoenzymatic assays to detect specific hybrids formed in solution. System 2, using a double sandwich enzyme-linked immunosorbent assay, and system 1, using a biotinylated probe, proved to be very effective. The method using biotin-streptavidin complex, however, resulted in a higher background signal. System 2 described here, using unlabeled probe, was more effective. This strategy allowed the detection of as little as 2.5 pg target RNA from pure culture and 500 cells from inoculated meat homogenate, even in the presence of other contaminating bacteria. The assay was more sensitive and could be completed within 3 h, as opposed to several days when conventional culture methods were used.     

Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes

Aims: Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi‐copy gene regions of Escherichia coli. Methods and Results: A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxonomic specificity and sensitivity of qPCR assays targeting the rodA gene (rodA984) and two regions of the multi‐copy 23S ribosomal RNA gene (EC23S and EC23S857). Experimental analyses of 28 culture collection strains representing E. coli and 21 related non‐target species indicated that the uidA405 and rodA984 assays were both 100% specific for E. coli while the EC23S assay was only 29% specific. The EC23S857 assay was only 95% specific due to detection of E. fergusonii. The uidA405, rodA984, EC23S and EC23S857 assays were 85%, 85%, 100% and 86% sensitive, respectively, in detecting 175 presumptive E. coli culture isolates from fresh, marine and waste water samples. In analyses of DNA extracts from 32 fresh, marine and waste water samples, the rodA984, EC23S and EC23S857 assays detected mean densities of target sequences at ratios of approximately 1 : 1, 243 : 1 and 6 : 1 compared with the mean densities detected by the uidA405 assay. Conclusions: The EC23S assay was less specific for E. coli, whereas the rodA984 and EC23S857 assay taxonomic specificities and sensitivities were similar to those of the uidA405 gene assay. Significance and Impact: The EC23S857 assay has a lower limit of detection for E. coli cells than the uidA405 and rodA984 assays due to its multi‐copy gene target and therefore provides greater analytical sensitivity in monitoring for these faecal pollution indicators in environmental waters by qPCR methods.     

High-flux isobutanol production using engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>: a bioreactor study with in situ product removal

Promising approaches to produce higher alcohols, e.g., isobutanol, using Escherichia coli have been developed with successful results. Here, we translated the isobutanol process from shake flasks to a 1-L bioreactor in order to characterize three E. coli strains. With in situ isobutanol removal from the bioreactor using gas stripping, the engineered E. coli strain (JCL260) produced more than 50 g/L in 72 h. In addition, the isobutanol production by the parental strain (JCL16) and the high isobutanol-tolerant mutant (SA481) were compared with JCL260. Interestingly, we found that the isobutanol-tolerant strain in fact produced worse than either JCL16 or JCL260. This result suggests that in situ product removal can properly overcome isobutanol toxicity in E. coli cultures. The isobutanol productivity was approximately twofold and the titer was 9% higher than n-butanol produced by Clostridium in a similar integrated system.     

PHYLOGENETIC PLACEMENT OF BOTRYOCOCCUS BRAUNII (TREBOUXIOPHYCEAE) AND BOTRYOCOCCUS SUDETICUS ISOLATE UTEX 2629 (CHLOROPHYCEAE)1

The phylogenetic placement of four isolates of Botryococcus braunii Kützing and of Botryococcus sudeticus Lemmermann isolate UTEX 2629 was investigated using sequences of the nuclear small subunit (18S) rRNA gene. The B. braunii isolates represent the A (two isolates), B, and L chemical races. One isolate of B. braunii (CCAP 807/1; A race) has a group I intron at Escherichia coli position 1046 and isolate UTEX 2629 has group I introns at E. coli positions 516 and 1512. The rRNA sequences were aligned with 53 previously reported rRNA sequences from members of the Chlorophyta, including one reported for B. braunii (Berkeley strain). Phylogenetic trees were constructed using distance, weighted maximum parsimony, and maximum likelihood, and their reliability was estimated using bootstrap analysis for distance and parsimony and Bayesian inference for likelihood. All methods showed, with high bootstrap or credibility support, that the four isolates of B. braunii form a monophyletic group whose closest relatives are in the genus Choricystis in the Trebouxiophyceae, whereas the previously reported B. braunii sequence is from a member of the Chlamydomonadales in the Chlorophyceae and isolate UTEX 2629 is a member of the Sphaeropleales in the Chlorophyceae. Polyphyly of these sequences was confirmed by Kishino‐Hasegawa tests on artificial trees in which sequences were moved to a single lineage.