DNA from Buccal Swabs Suitable for High-Throughput SNP Multiplex Analysis

We sought a convenient and reliable method for collection of genetic material that is inexpensive and noninvasive and suitable for self-collection and mailing and a compatible, commercial DNA extraction protocol to meet quantitative and qualitative requirements for high-throughput single nucleotide polymorphism (SNP) multiplex analysis on an automated platform. Buccal swabs were collected from 34 individuals as part of a pilot study to test commercially available buccal swabs and DNA extraction kits. DNA was quantified on a spectrofluorometer with Picogreen dsDNA prior to testing the DNA integrity with predesigned SNP multiplex assays. Based on the pilot study results, the Catch-All swabs and Isohelix buccal DNA isolation kit were selected for our high-throughput application and extended to a further 1140 samples as part of a large cohort study. The average DNA yield in the pilot study (n=34) was 1.94 μg ± 0.54 with a 94% genotyping pass rate. For the high-throughput application (n=1140), the average DNA yield was 2.44 μg ± 1.74 with a ≥93% genotyping pass rate. The Catch-All buccal swabs are a convenient and cost-effective alternative to blood sampling. Combined with the Isohelix buccal DNA isolation kit, they provided DNA of sufficient quantity and quality for high-throughput SNP multiplex analysis.     

A Simple Method of Genomic DNA Extraction from Human Samples for PCR-RFLP Analysis

Isolation of DNA from blood and buccal swabs in adequate quantities is an integral part of forensic research and analysis. The present study was performed to determine the quality and the quantity of DNA extracted from four commonly available samples and to estimate the time duration of the ensuing PCR amplification. Here, we demonstrate that hair and urine samples can also become an alternate source for reliably obtaining a small quantity of PCR-ready DNA. We developed a rapid, cost-effective, and noninvasive method of sample collection and simple DNA extraction from buccal swabs, urine, and hair using the phenol-chloroform method. Buccal samples were subjected to DNA extraction, immediately or after refrigeration (4–6°C) for 3 days. The purity and the concentration of the extracted DNA were determined spectrophotometerically, and the adequacy of DNA extracts for the PCR-based assay was assessed by amplifying a 1030-bp region of the mitochondrial D-loop. Although DNA from all the samples was suitable for PCR, the blood and hair samples provided a good quality DNA for restriction analysis of the PCR product compared with the buccal swab and urine samples. In the present study, hair samples proved to be a good source of genomic DNA for PCR-based methods. Hence, DNA of hair samples can also be used for the genomic disorder analysis in addition to the forensic analysis as a result of the ease of sample collection in a noninvasive manner, lower sample volume requirements, and good storage capability.     

A family exhibiting heteroplasmy in the human mitochondrial DNA control region reveals both somatic mosaicism and pronounced segregation of mitotypes

A family exhibiting heteroplasmy at position 16 355 in hypervariable region I of the human mtDNA control region has been identified. This family consists of a mother, daughter, and son. DNA samples extracted from blood stains, buccal swabs, and hairs from these individuals were amplified by PCR and sequenced utilizing fluoresence-labeled dye terminator chemistry in an automated DNA sequencer. In both the daughter and mother, heteroplasmy was observed in DNA extracted from blood stains, buccal swabs, and hairs. In the blood stains, the proportion of cytosine was greater than thymine in both individuals. Buccal swab extracts showed a more balanced contribution from the two nucleotides. Telogenic hair root and hair shaft samples exhibited a wide range of nucleotide contributions at this position, from predominately cytosine in some samples to predominately thymine in others. The apparent stochastic segregation of mitotypes in hair samples is discussed from a forensic viewpoint, and the mechanism of mtDNA heteroplasmy is considered. Received: 6 November 1996 / Accepted: 13 February 1997     

Validation of a cheap and simple nondestructive method for obtaining AFLPs and DNA sequences (mitochondrial and nuclear) in amphibians

The use of nondestructive methods for obtaining DNA from amphibians (e.g. buccal swabs) allows genetic studies to be performed without affecting the survival of the studied individuals. In this study, we compared two methods of nondestructive DNA sampling, buccal swabs and interdigital membrane or toe‐clipping, in several amphibian species of different size: Rhinella spinulosa, R. atacamensis, six species of the genus Telmatobius and Pleurodema thaul. We evaluated the integrity of the DNA extracted by sequencing fragments of mitochondrial and nuclear genes and by generating amplified fragment length polymorphisms markers (AFLPs). In all cases, we obtained an adequate amount of DNA (mean range 55–298 ng/μL). We obtained identical DNA sequences from buccal swab and interdigital membrane/toe‐clip for all individuals. The differences in the coding of AFLP markers between the tissues were similar to those reported for replicas of the same type of sample in similar analyses in other species of amphibians. In conclusion, the use of buccal swabs is a trustworthy and inexpensive method to obtain DNA for mitochondrial and nuclear sequencing and AFLP analyses. Given the types of markers evaluated, buccal swabs may be used for phylogenetic, phylogeographic and population genetic studies, even in small amphibians (<33 mm).     

Comparison between two techniques for endometrial swab culture and between biopsy and culture in barren mares

The endometria of 77 barren mares was swabbed simultaneously using a swab guarded with a single cannula and distal, gelatin capsule (completely guarded swab - CGS) and a partially guarded swab (PGS) with an open cannula. Sheep blood (5%) agar plates were inoculated with each swab, while MacConkey's agar plates were inoculated with the swabs from 44 mares. The presence of bacterial or fungal growth was determined after 24 and 48 hours of aerobic incubation at 37 C. Organisms present were identified, counted, and categorized as saprophytic or pathogenic flora. The endometria of all mares were biopsied immediately following swabbing. Histologic evidence of inflammation in biopsy specimens was classified as (1) none, (2) slight, discrete, focal, and (3) slight or moderate, diffuse, widespread infiltration of inflammatory cells. The number of inflammatory cells migrating through the luminal epithelium was counted and averaged. There were significantly fewer CGS than PGS cultures that yielded growth at 24 and 48 hours of incubation after being streaked on blood agar and MacConkey's agar plates. There were fewer pathogenic bacterial or fungal colonies present at 48 hours of incubation on blood agar plates after being streaked with CGS as compared to PGS. There were no differences in the number of pathogenic bacterial or fungal colonies present at 24 hours of incubation on blood agar or at 24 and 48 hours of incubation on MacConkey's agar plates. There was no correlation between CGS or PGS culture of pathogens and severity of histologic inflammation. There was a positive correlation between culture of pathogens and number of inflammatory cells migrating through the luminal epithelium.     

Cloacal and buccal swabs are a reliable source of DNA for microsatellite genotyping of reptiles

In this study, a minimally invasive method for DNA sampling of reptiles and amphibians using cloacal and buccal swabs is described. High molecular weight DNA was isolated from the swabs, which were collected from tuatara (Sphenodon punctatus), and stored in 70% ethanol at room temperature for approximately 1 week. Amplification of mitochondrial and microsatellite DNA loci was successful from both cloacal and buccal swabs, and in all cases the genotypes matched those obtained from blood samples. These results show that cloacal and/or buccal swabbing is a useful alternative to blood sampling and toe clipping for genetic studies on reptiles. This method is rapid, inexpensive and easy to implement in field situations.     

Detecting the immune system response of a 500 year-old inca mummy

Disease detection in historical samples currently relies on DNA extraction and amplification, or immunoassays. These techniques only establish pathogen presence rather than active disease. We report the first use of shotgun proteomics to detect the protein expression profile of buccal swabs and cloth samples from two 500-year-old Andean mummies. The profile of one of the mummies is consistent with immune system response to severe pulmonary bacterial infection at the time of death. Presence of a probably pathogenic Mycobacterium sp. in one buccal swab was confirmed by DNA amplification, sequencing, and phylogenetic analyses. Our study provides positive evidence of active pathogenic infection in an ancient sample for the first time. The protocol introduced here is less susceptible to contamination than DNA-based or immunoassay-based studies. In scarce forensic samples, shotgun proteomics narrows the range of pathogens to detect using DNA assays, reducing cost. This analytical technique can be broadly applied for detecting infection in ancient samples to answer questions on the historical ecology of specific pathogens, as well as in medico-legal cases when active pathogenic infection is suspected.     

Skin swabbing as a new efficient DNA sampling technique in amphibians, and 14 new microsatellite markers in the alpine newt (Ichthyosaura alpestris)

This study introduces a novel DNA sampling method in amphibians using skin swabs. We assessed the relevancy of skin swabs relevancy for genetic studies by amplifying a set of 17 microsatellite markers in the alpine newt Ichthyosaura alpestris, including 14 new polymorphic loci, and a set of 11 microsatellite markers in Hyla arborea, from DNA collected with buccal swabs (the standard swab method), dorsal skin swabs and ventral skin swabs. We tested for quality and quantity of collected DNA with each method by comparing electrophoresis migration patterns. The consistency between genotypes obtained from skin swabs and buccal swabs was assessed. Dorsal swabs performed better than ventral swabs in both species, possibly due to differences in skin structure. Skin swabbing proved to be a useful alternative to buccal swabbing for small or vulnerable animals: by drastically limiting handling, this method may improve the trade-off between the scientific value of collected data, individual welfare and species conservation. In addition, the 14 new polymorphic microsatellites for the alpine newt will increase the power of genetic studies in this species. In four populations from France (n=19-25), the number of alleles per locus varied from 2 to 16 and expected heterozygosities ranged from 0.04 to 0.91. Presence of null alleles was detected in two markers and two pairs displayed gametic disequilibrium. No locus appeared to be sex-linked.     

Quantitative assessment of vaginal microflora during use of tampons of various compositions

Although the effect of vaginal tampons on the microbial flora during menstruation has recently been studied by several investigators, quantitative effects attributable to particular tampon fibers have received less attention. The purposes of the present study were (i) to determine and then to compare the effects of polyacrylate rayon tampons and viscose rayon tampons on the normal vaginal flora, (ii) to compare quantitative bacterial counts obtained from these tampons with those obtained from concomitant vaginal swabs, and (iii) to determine whether either of these tampon types alters the vaginal microflora when compared with the microflora in the same women using all-cotton tampons or external catamenial pads. Tampon and swab samples were obtained at predetermined times from 18 women for an average of seven menstrual cycles. Samples consisting of swabs from women wearing menstrual pads were compared with swab and tampon samples taken at predetermined times during the menstrual cycle from women using cotton, polyacrylate rayon, or viscose rayon tampons. Samples were analyzed for total aerobic, facultative, and anaerobic bacterial counts. Statistical evaluation of the results indicated that, on the whole, total bacterial counts decreased during menstruation and that the numbers of bacteria in tampons tended to be lower than those in swab samples taken at the same time. The tampon type had little effect on the vaginal microflora.     

Cost-effective analysis of genotyping using oral cells in the geriatric population

We evaluated the cost-effectiveness of using buccal swab brushes in comparison with blood samples for obtaining DNA for large epidemiological studies of the elderly population. The data reported here are from the third phase of the Integral Study of Depression among the Elderly in Mexico City's Mexican Institute of Social Security, conducted in 2007. The total cost of the two procedures was determined. The measurement of effectiveness was the quality and quantity of DNA measured in ng/μL and the use of this DNA for the determination of apolipoprotein E (APO E) polymorphism by PCR. Similar rates of amplification were obtained with the two techniques. The cost of the buccal swab brushes, including sample collection and DNA extraction, was US$16.63, compared to the cost per blood sample of US$23.35. Using the buccal swab, the savings was US$6.72 per patient (P < 0.05). The effectiveness was similar. Quantity and quality of DNA obtained were similar for the oral and blood procedures, demonstrating that the swab brush technique offers a feasible alternative for large-scale epidemiological studies.     

Quantitative assessment of vaginal microflora during use of tampons of various compositions.

Although the effect of vaginal tampons on the microbial flora during menstruation has recently been studied by several investigators, quantitative effects attributable to particular tampon fibers have received less attention. The purposes of the present study were (i) to determine and then to compare the effects of polyacrylate rayon tampons and viscose rayon tampons on the normal vaginal flora, (ii) to compare quantitative bacterial counts obtained from these tampons with those obtained from concomitant vaginal swabs, and (iii) to determine whether either of these tampon types alters the vaginal microflora when compared with the microflora in the same women using all-cotton tampons or external catamenial pads. Tampon and swab samples were obtained at predetermined times from 18 women for an average of seven menstrual cycles. Samples consisting of swabs from women wearing menstrual pads were compared with swab and tampon samples taken at predetermined times during the menstrual cycle from women using cotton, polyacrylate rayon, or viscose rayon tampons. Samples were analyzed for total aerobic, facultative, and anaerobic bacterial counts. Statistical evaluation of the results indicated that, on the whole, total bacterial counts decreased during menstruation and that the numbers of bacteria in tampons tended to be lower than those in swab samples taken at the same time. The tampon type had little effect on the vaginal microflora.     

An Improved Field Method to Obtain DNA for Individual Identification From Wolf Scat

ABSTRACT Sampling of feces for genetic studies of wild populations can be problematic because of the low quality and quantity of template DNA obtained. We used cotton swabs in the field to isolate the mucous layer on the surface of fresh wolf (Canis lupus, C. lycaon, and their hybrids) scats followed by immediate preservation, and compared microsatellite genotyping of DNA from these fresh field swabs (FS) to that of previously frozen laboratory swabs (LS). In single polymerase chain reactions (PCRs) of 2 multiplexes, amplification at 8 loci was higher in the FS samples (FS = 50%, LS = 15%; P = 0.02) because proportion, quantity, and quality of large fragment wolf nuclear DNA from these samples was greater (2.5–25%, 6.25–62.5 ng/swab, 35% amplified at 1,000 base pairs [bp]) than from the LS samples (1.9%–10%, 4.7–25 ng/swab, 10% amplified at 1,000 bp). Paired blood and fresh field-swabbed samples had identical genotypes. In 84 multiplex PCRs we found no evidence of allelic dropout associated with low template quality or quantity. We conclude that field swabbing of fresh wolf scat facilitates field storage and reduces the need for multiple amplifications at single microsatellite loci, thereby reducing the genotyping costs for wildlife projects that use noninvasive samples.     

Swab Sample Transfer for Point-Of-Care Diagnostics: Characterization of Swab Types and Manual Agitation Methods

Background

The global need for disease detection and control has increased effort to engineer point-of-care (POC) tests that are simple, robust, affordable, and non-instrumented. In many POC tests, sample collection involves swabbing the site (e.g., nose, skin), agitating the swab in a fluid to release the sample, and transferring the fluid to a device for analysis. Poor performance in sample transfer can reduce sensitivity and reproducibility.

Methods

In this study, we compared bacterial release efficiency of seven swab types using manual-agitation methods typical of POC devices. Transfer efficiency was measured using quantitative PCR (qPCR) for Staphylococcus aureus under conditions representing a range of sampling scenarios: 1) spiking low-volume samples onto the swab, 2) submerging the swab in excess-volume samples, and 3) swabbing dried sample from a surface.

Results

Excess-volume samples gave the expected recovery for most swabs (based on tip fluid capacity); a polyurethane swab showed enhanced recovery, suggesting an ability to accumulate organisms during sampling. Dry samples led to recovery of ∼20–30% for all swabs tested, suggesting that swab structure and volume is less important when organisms are applied to the outer swab surface. Low-volume samples led to the widest range of transfer efficiencies between swab types. Rayon swabs (63 µL capacity) performed well for excess-volume samples, but showed poor recovery for low-volume samples. Nylon (100 µL) and polyester swabs (27 µL) showed intermediate recovery for low-volume and excess-volume samples. Polyurethane swabs (16 µL) showed excellent recovery for all sample types. This work demonstrates that swab transfer efficiency can be affected by swab material, structure, and fluid capacity and details of the sample. Results and quantitative analysis methods from this study will assist POC assay developers in selecting appropriate swab types and transfer methods.     

Evaluation of a New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate Surfaces

Inanimate surfaces are regarded as key vehicles for the spread of human norovirus during outbreaks. ISO method 15216 involves the use of cotton swabs for environmental sampling from food surfaces and fomites for the detection of norovirus genogroup I (GI) and GII. We evaluated the effects of the virus drying time (1, 8, 24, or 48 h), swab material (cotton, polyester, rayon, macrofoam, or an antistatic wipe), surface (stainless steel or a toilet seat), and area of the swabbed surface (25.8 cm² to 645.0 cm²) on the recovery of human norovirus. Macrofoam swabs produced the highest rate of recovery of norovirus from surfaces as large as 645 cm². The rates of recovery ranged from 2.2 to 36.0% for virus seeded on stainless-steel coupons (645.0 cm²) to 1.2 to 33.6% for toilet seat surfaces (700 cm²), with detection limits of 3.5 log₁₀ and 4.0 log₁₀ RNA copies. We used macrofoam swabs to collect environmental samples from several case cabins and common areas of a cruise ship where passengers had reported viral gastroenteritis symptoms. Seventeen (18.5%) of 92 samples tested positive for norovirus GII, and 4 samples could be sequenced and had identical GII.1 sequences. The viral loads of the swab samples from the cabins of the sick passengers ranged from 80 to 31,217 RNA copies, compared with 16 to 113 RNA copies for swab samples from public spaces. In conclusion, our swab protocol for norovirus may be a useful tool for outbreak investigations when no clinical samples are available to confirm the etiology.     

Treatment of streptococcal pharyngitis with amoxycillin once a day.

OBJECTIVE--To evaluate treatment of group A beta haemolytic streptococcal pharyngitis with amoxycillin once daily compared with phenoxymethylpenicillin three or four times a day. DESIGN--Randomised controlled study of consecutive patients presenting with symptoms suggestive of group A beta haemolytic streptococcal pharyngitis in whom culture of a throat swab yielded positive results. SETTING--Five family medicine practices. SUBJECTS--157 patients aged over 3 years who required treatment with antibiotics. MAIN OUTCOME MEASURES--Clinical response, bacteriological response, days at work and school lost, and compliance. RESULTS--During the period of the study 393 patients presented with symptoms suggesting streptococcal pharyngitis; 157 of them had throat swabs that yielded positive results on culture. Eighty two were treated with phenoxymethylpenicillin and 75 with amoxycillin. No difference was observed in the clinical response, days at work and school lost (139 days for 64 patients taking phenoxymethylpenicillin v 100 days for 57 patients taking amoxycillin; p > 0.2), or residual positive cultures after two days (6 (7.3%) v 3 (4%); p > 0.5). A significant difference in the bacteriological response was found after 14 days (5 (6.1%) v 0; p < 0.04) with no positive cultures observed in the amoxycillin group. CONCLUSIONS--These findings support the hypothesis that amoxycillin once daily is as effective as phenoxymethylpenicillin in the treatment of group A beta haemolytic streptococcal pharyngitis.     

From the Field to the Lab: Best Practices for Field Preservation of Bat Specimens for Molecular Analyses

Studies in molecular ecology depend on field-collected samples for genetic information, and the tissue sampled and preservation conditions strongly affect the quality of the DNA obtained. DNA yields from different tissue types have seldom been compared, and the relative performance of storage media has never been directly tested, even though these media may influence DNA degradation under field conditions. We analyzed DNA yield from buccal swabs and wing punches harvested from live bats using nucleic acid quantification as well as quantitative PCR for a single-copy nuclear locus. We also compared DNA yields from wing tissue preserved in three media: ethanol, NaCl-saturated dimethyl sulfoxide (DMSO), and silica desiccant. Wing punches yielded more total DNA than did buccal swabs, and wing tissues preserved in silica beads yielded significantly more total and nuclear DNA than those preserved in DMSO or ethanol. These results show that tissue type and preservation media strongly influence the quantity of DNA obtained from non-lethal genetic samples, and based on these effects we provide recommendations for field collection of tissues for genetic analyses.     

DNA isolation of mucus from Salmo trutta (Linnaeus, 1758) and Thymallus thymallus (Linnaeus, 1758) as an alternative method to conventional fin‐clipping

The first objective of the actual project was to investigate if mucus from Salmo trutta (Linnaeus, 1758) and Thymallus thymallus (Linnaeus, 1758) is suitable for DNA extraction. In the second step, it was analyzed if the avoidance of environmentally hazardous chemicals could lead to sufficiently pure DNA isolation. Finally, it was examined if samples taken from different fish body parts yielded different grades of purity of the extracted DNA. Mucus was collected from 552 individuals of S. truttaand T. thymallusfrom 2012 to 2015. DNA was extracted with two different kits (E.Z.N.A.^® Insect DNA Kit and my‐Budget DNA Mini Kit) and two different swabs (my‐Budget roughened laminated sterile cotton swabs and common autoclaved cotton swabs). The results showed that the my‐Budget DNA Mini Kit in combination with the my‐Budget roughened laminated sterile cotton swabs was the most suitable method. It is a non‐destructive and swift technique that results in the concentration of DNA being high and pure.     

Pros and cons of external swabbing of amphibians for genetic analyses

Non-invasive DNA sampling is an important tool in amphibian conservation. Buccal swabs are nowadays replacing the wounding toe-clipping method. Skin and cloaca swabbing are even less invasive and easier to handle than buccal swabbing, but could result in contaminations of genetic material. Therefore, we test if external skin and cloaca swabs are as reliable as buccal swabs for genetic analysis of amphibians. We analysed eight microsatellite loci for the common frog (Rana temporaria, Linnaeus 1758) and compared genotyping results for buccal, skin and cloaca swabs regarding allelic dropouts and false alleles. Furthermore, we compared two DNA extraction methods regarding efficiency and cost. DNA quality and quantity (amplification success, genotyping error rate, in nanogram per microlitre) were comparable among DNA sources and extraction methods. However, skin and cloaca samples exhibited high degrees of contamination with foreign individuals, which was due to sample collection during mating season. Here, we established a simple low budget procedure to receive DNA of amphibians avoiding stressful buccal swabbing or harmful toe clipping. However, the possibility of contaminations of external swabs has to be considered.     

Comparison of proteases in DNA extraction via quantitative polymerase chain reaction

We compared four proteases in the QIAamp DNA Investigator Kit (Qiagen) to extract DNA for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate alternate proteases for improved DNA recovery as compared with proteinase K for forensic, biochemical research, genetic paternity and immigration, and molecular diagnostic purposes. The Quantifiler Kit TaqMan quantitative PCR assay was used to measure the recovery of DNA from human blood, semen, buccal cells, breastmilk, and earwax in addition to low-template samples, including diluted samples, computer keyboard swabs, chewing gum, and cigarette butts. All methods yielded amplifiable DNA from all samples.     

High fractions of exogenous DNA in human buccal samples reduce the quality of large-scale genotyping

Buccal cell samples are considered a reliable source of DNA for genotyping studies. However, a potential drawback is the presence of exogenous DNA that is coextracted with human genomic DNA. A set of saliva and cheek swab samples, in which the fraction of human DNA varies from 10 to 96%, was genotyped using the Affymetrix Mapping 500 K Array. Samples containing less than 30% human DNA performed poorly in terms of accuracy and reliability. Therefore, we recommend quantitating the amount of human DNA in buccal samples to be used for large-scale genotyping and eliminating samples with less than 30% human DNA.