Reliable microsatellite genotyping of dolphin DNA from faeces

Noninvasive samples have proved useful in genotyping studies of free‐ranging mammals. However, potential genotyping errors associated with such samples dictate the need for validation studies. This pilot study demonstrates the use of dolphin faeces in multilocus microsatellite genotyping studies. An empirical approach to calculating the rate of genotyping error was applied to data from matched pairs of blood or tissue and faecal samples from both captive and wild bottlenose dolphins. Microsatellite genotypes were assigned to dolphin faecal extracts with greater than 95% confidence by using a multiple tube approach, and at least two independent replicate genotypings.     

Factors affecting the amount of genomic DNA extracted from ape faeces and the identification of an improved sample storage method

Abstract Genetic analysis using noninvasively collected samples such as faeces continues to pose a formidable challenge because of unpredictable variation in the extent to which usable DNA is obtained. We investigated the influence of multiple variables on the quantity of DNA extracted from faecal samples from wild mountain gorillas and chimpanzees. There was a small negative correlation between temperature at time of collection and the amount of DNA obtained. Storage of samples either in RNAlater solution or dried using silica gel beads produced similar results, but significantly higher amounts of DNA were obtained using a novel protocol that combines a short period of storage in ethanol with subsequent desiccation using silica.     

An optimisation approach to increase DNA amplification success of otter faeces

Faeces have proved to be a suitable non-invasive DNA source for microsatellite analysis in wildlife research. For the success of such studies it is essential to obtain the highest possible PCR amplification success rate. These rates are still relatively low in most carnivorous species, especially in the otter (Lutra lutra). We therefore optimised the entire microsatellite genotyping process by combining our findings with results from previous studies to gain a high rate of reliable genotypes. We investigated the influence of otter faecal quality in relation to the quantity of slimy secretions and three levels of storage periods at ?20°C on amplification success. Further, we tested the cost-effective and time-saving Chelex extraction method against the profitable QIAamp^® DNA Stool Kit (Qiagen), and compared three PCR methods - a standard single-step PCR protocol, a single-locus two-step PCR procedure and a multiplex two-step PCR procedure - regarding success rate and genotyping errors. The highest amplification success rate (median: 94%; mean: 78%) was achieved using faecal samples consisting only of jelly extracted with the QIAamp^® DNA Stool Mini Kit (Qiagen) immediately after collection and amplified following the time and cost efficient multiplex two-step PCR protocol. The two-step procedure, also referred to as pre-amplification approach, turned out to be the main improvement as it increases amplification success about 11% and reduces genotyping errors about 53%, most notably allelic dropouts.     

Extensive human DNA contamination in extracts from ancient dog bones and teeth

Ancient DNA (aDNA) sequences, especially those of human origin, are notoriously difficult to analyze due to molecular damage and exogenous DNA contamination. Relatively few systematic studies have focused on this problem. Here we investigate the extent and origin of human DNA contamination in the most frequently used sources for aDNA studies, that is, bones and teeth from museum collections. To distinguish contaminant DNA from authentic DNA we extracted DNA from dog (Canis familiaris) specimens. We monitored the presence of a 148-bp human-specific and a 152-bp dog-specific mitochondrial DNA (mtDNA) fragment in DNA extracts as well as in negative controls. The total number of human and dog template molecules were quantified using real-time polymerase chain reaction (PCR), and the sequences were characterized by amplicon cloning and sequencing. Although standard precautions to avoid contamination were taken, we found that all samples from the 29 dog specimens contained human DNA, often at levels exceeding the amount of authentic ancient dog DNA. The level of contaminating human DNA was also significantly higher in the dog extracts than in the negative controls, and an experimental setup indicated that this was not caused by the carrier effect. This suggests that the contaminating human DNA mainly originated from the dog bones rather than from laboratory procedures. When cloned, fragments within a contaminated PCR product generally displayed several different sequences, although one haplotype was often found in majority. This leads us to believe that recognized criteria for authenticating aDNA cannot separate contamination from ancient human DNA the way they are presently used.     

Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.     

Quantitative polymerase chain reaction analysis of DNA from noninvasive samples for accurate microsatellite genotyping of wild chimpanzees (Pan troglodytes verus)

Noninvasive samples are useful for molecular genetic analyses of wild animal populations. However, the low DNA content of such samples makes DNA amplification difficult, and there is the potential for erroneous results when one of two alleles at heterozygous microsatellite loci fails to be amplified. In this study we describe an assay designed to measure the amount of amplifiable nuclear DNA in low DNA concentration extracts from noninvasive samples. We describe the range of DNA amounts obtained from chimpanzee faeces and shed hair samples and formulate a new efficient approach for accurate microsatellite genotyping. Prescreening of extracts for DNA quantity is recommended for sorting of samples for likely success and reliability. Repetition of results remains extensive for analysis of microsatellite amplifications beginning from low starting amounts of DNA, but is reduced for those with higher DNA content.     

A novel method for collection and preservation of faeces for genetic studies

Quantitative and qualitative measurements of DNA were used to compare faecal sample storage in ethanol and silica with a novel method (two‐step) in which samples are soaked in ethanol and then desiccated with silica. Silica‐preserved samples had the lowest DNA concentrations. The two‐step method yielded significantly more DNA in high quality samples (average DNA concentrations > 100 pg/µL with all storage methods). However, for lower quality samples, the ethanol and two‐step methods performed similarly. The amounts and rates of sample degradation were not strongly affected by storage method and neither was the percentage of target DNA (< 1%) obtained from the samples.     

A new 2CTAB/PCI method improves DNA amplification success from faeces of Mediterranean (Barbary macaques) and tropical (lowland gorillas) primates

Animal genomic DNA extracts of sufficient quality to address questions about population biology or behavioural ecology can be obtained from faeces when adequate extraction procedures are used. The presence of PCR inhibitors in extract products appears generally the main factor limiting DNA amplification success. We compared DNA amplification success from faeces of a tropical primate (western lowland gorilla, Gorilla g. gorilla) and a Mediterranean primate (Barbary macaque, Macaca sylvanus) between a standardized extraction technique widely used in animals (QIAamp^® stool kit), a technique mainly used in plant species (CTAB) and a new protocol (2CTAB/PCI). Amplification success varied from 51% to 97%, the highest success being reached with the 2CTAB/PCI protocol in both species.     

Similar estimates of population genetic composition and sex ratio derived from carcasses and faeces of Eurasian otter Lutra lutra

Collecting faeces is viewed as a potentially efficient way to sample elusive animals. Nonetheless, any biases in estimates of population composition associated with such sampling remain uncharacterized. The goal of this study was to compare estimates of genetic composition and sex ratio derived from Eurasian otter Lutra lutra spraints (faeces) with estimates derived from carcasses. Twenty per cent of 426 wild-collected spraints from SW England yielded composite genotypes for 7-9 microsatellites and the SRY gene. The expected number of incorrect spraint genotypes was negligible, given the proportions of allele dropout and false allele detection estimated using paired blood and spraint samples of three captive otters. Fifty-two different spraint genotypes were detected and compared with genotypes of 70 otter carcasses from the same area. Carcass and spraint genotypes did not differ significantly in mean number of alleles, mean unbiased heterozygosity or sex ratio, although statistical power to detect all but large differences in sex ratio was low. The genetic compositions of carcass and spraint genotypes were very similar according to confidence intervals of theta and two methods for assigning composite genotypes to groups. A distinct group of approximately 11 carcass and spraint genotypes was detected using the latter methods. The results suggest that spraints can yield unbiased estimates of population genetic composition and sex ratio.     

Factors affecting success of PCR amplification of microsatellite loci from otter faeces

We investigated the effect of multiple variables on the amplification success rate of microsatellite DNA extracted from faeces of wild Eurasian otters. The success rate was affected by (i) type of sample, with higher success rates in anal jelly samples than faeces, and (ii) temperature, with a negative effect of increased temperature at time of collection. To increase the effectiveness of microsatellite genotyping of otter faeces, we recommend collecting samples in cold months and early in the morning, preferably in a frozen state, and the collection of anal jelly samples, or the jelly part from faeces, whenever possible.     

DNA extraction from bovine faeces: current status and future trends

There is an increasing interest in the detection and enumeration of micro‐organisms pathogenic for human and present in bovine faeces. This interest is because pollution of the environment by animal faeces may affect the safety of food and of drinking or recreational water. Detection and quantification of microbial pathogens carried out using DNA extracted from the faecal matrix are affected by the quality and the quantity of the DNA extracts, which are critical factors that limit the accuracy and sensitivity of molecular studies. This review compares published methods on DNA extraction from bovine faeces, focusing on the extent to which the success of DNA amplification is affected by issues related to the faeces. Following a general discussion on the DNA extraction methods used for faeces, we focus particularly on issues related to the faecal environment itself. The objective is to identify information that can be used to improve the sensitivity of those PCR methods used after direct DNA extraction.     

Effects of post-ingestion and physical conditions on PCR amplification of host blood meal DNA in mosquitoes

The effects of post-ingestion and physical conditions under which killed mosquitoes are stored on the success of detecting blood meal DNA of Anopheles stephensi and Culex quinquefasiatus were investigated by polymerase chain reaction (PCR) amplification at the human mitochondrial DNA cytochrome b (cytB) gene. Host DNA extracted from the blood meal up to 33 h post-ingestion in both species acts as an efficient template for PCR amplification. However, more DNA concentration is needed for meals digested for a longer time, and successful PCR amplification from meals digested for 36 h,dropped to a faint band. There were no differences between PCR success rate for samples stored at +4 or -20 degrees C, but less successful products were observed in samples kept at 4 degrees C for the periods longer than 30 h digestion. The results of this study are important for conducting malaria epidemiological studies that provide information about the degree of contact between human hosts and mosquito vectors, impact of vector controls such as bed nets and repellents, and the transmission dynamics of human malaria and other vector-borne diseases.     

Bryophyte DNA sequences from faeces of an arctic herbivore, barnacle goose (Branta leucopsis)

We tested DNA extraction methods and PCR conditions for the amplification of bryophyte DNA from barnacle goose (Branta leucopsis) faeces collected from Spitsbergen (Svalbard). Both the Qiagen stool kit and a silica-based extraction method received sufficient DNA from fresh and older droppings, as indicated by successful amplification of the plastid psbA-trnH spacer. Standard Taq polymerase outperformed two hot start polymerases. Sequencing of cloned PCR products revealed at least ten moss and two angiosperm sequences. This first example of identifying bryophyte DNA from faeces will allow analysing moss diets of arctic herbivores with a DNA barcoding approach.     

DNA ligases from thermophilic bacteria enhance PCR amplification of long DNA sequences

Bacterial NAD-dependent Taq and Tth DNA ligases are capable of significantly increasing the yield of long PCR products when the amplification is carried out using bacterial family A DNA polymerases, e.g. Taq or Tth DNA polymerases, or with enzymatic blends containing these polymerases. We also show that Taq and Tth DNA ligases improve the results of PCR in the absence of NAD and therefore in the absence of DNA ligase activity. These observations suggest that bacterial DNA ligases can interact with these DNA polymerases, presumably as accessory proteins, thereby enhancing the efficiency of DNA polymerization. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 5, pp. 685–690.     

PCR detection of Brachyspira aalborgi and Brachyspira pilosicoli in human faeces

Previously-developed PCR protocols specific for the 16S rRNA gene of the intestinal spirochaetes Brachyspira aalborgi and Brachyspira pilosicoli were adapted for the detection of these species in human faeces, following DNA extraction and purification using mini-prep columns. The limits of detection in seeded faeces for B. aalborgi and B. pilosicoli respectively were 2x10(2) and 7x10(3) cells per PCR reaction, equivalent to 5x10(4) and 1x10(5) cells per g of faeces. The PCR techniques were applied to faecal samples from two patients with histological evidence of intestinal spirochaetosis. In the first patient, in whom B. aalborgi had been identified by 16S rDNA PCR from colonic biopsies, a positive amplification for B. aalborgi only was obtained from the faeces. The organism could not be isolated from these faeces. In the second patient, both colonic biopsies and faeces were PCR positive for B. pilosicoli only, and B. pilosicoli was isolated from the faeces. These new faecal PCR protocols should be valuable for future studies on the epidemiology of intestinal spirochaete infections in human populations, particularly as it is not currently possible to isolate B. aalborgi from faeces.     

基于数字PCR的不同品种鸭组织中线粒体与核DNA拷贝数差异研究

肉和肉制品是人类生活的重要营养来源,但近年来肉制品中发生的掺假使假事件屡见不鲜,使得肉品的质量安全问题已经成为全世界关注的热点话题。以核酸为目标的动物源鉴定是当前普遍使用的方法。在核酸检测中,常用线粒体基因或核基因作为靶标,缺乏统一标准。以绍兴鸭和北京鸭等不同品种及生鲜组织(鸭血、鸭胸肉、鸭肝、鸭皮、鸭心和鸭腿肉)为实验材料,提取DNA后利用微滴式数字PCR开展线粒体和核DNA拷贝数的比较研究,以两者拷贝数及其比值的变异系数为判定依据。结果显示,核DNA的拷贝数在不同品种鸭组织间相对稳定,且变异系数小于线粒体DNA,表明核DNA是开展鸭肉制品掺假定量检测的最适DNA来源。鸭腿肉中线粒体/核DNA拷贝数比值的变异系数最小,表明线粒体DNA作为靶基因的鸭肉掺假比例定量检测时,鸭腿肉来源的肉制品是最佳选择。     

Identification and quantification of viable Bifidobacterium breve strain Yakult in human faeces by using strain-specific primers and propidium monoazide

Aims: To develop a quick and accurate PCR‐based method to evaluate viable Bifidobacterium breve strain Yakult (BbrY) in human faeces. Methods and Results: The number of BbrY in faeces was detected by using strain‐specific quantitative real‐time PCR (qPCR) derived from a randomly amplified polymorphic DNA analysis. And using propidium monoazide (PMA) treatment, which combined a DNA‐intercalating dye for covalently linking DNA in dead cells and photoactivation, only viable BbrY in the faeces highly and significantly correlated with the number of viable BbrY added to faecal samples within the range of 10⁵–10⁹ cells per g of faeces was enumerated. After 11 healthy subjects ingested 10·7 log CFU of BbrY daily for 10 days, 6·9 (±1·5) log CFU g^?1 [mean (±SD)] of BbrY was detected in faeces by using strain‐specific transgalactosylated oligosaccharide–carbenicillin (T‐CBPC) selective agar medium. Viable BbrY detected by qPCR with PMA treatment was 7·5 (±1·0) log cells per g and the total number (viable and dead) of BbrY detected by qPCR without PMA treatment was 8·1 (±0·8) log cells per g. Conclusions: Strain‐specific qPCR with PMA treatment evaluated viable BbrY in faeces quickly and accurately. Significance and Impact of the Study: Combination of strain‐specific qPCR and PMA treatment is useful for evaluating viable probiotics and its availability in humans.     

Spiking of contemporary human template DNA with ancient DNA extracts induces mutations under PCR and generates nonauthentic mitochondrial sequences

Proof of authenticity is the greatest challenge in palaeogenetic research, and many safeguards have become standard routine in laboratories specialized on ancient DNA research. Here we describe an as-yet unknown source of artifacts that will require special attention in the future. We show that ancient DNA extracts on their own can have an inhibitory and mutagenic effect under PCR. We have spiked PCR reactions including known human test DNA with 14 selected ancient DNA extracts from human and nonhuman sources. We find that the ancient DNA extracts inhibit the amplification of large fragments to different degrees, suggesting that the usual control against contaminations, i.e., the absence of long amplifiable fragments, is not sufficient. But even more important, we find that the extracts induce mutations in a nonrandom fashion. We have amplified a 148-bp stretch of the mitochondrial HVRI from contemporary human template DNA in spiked PCR reactions. Subsequent analysis of 547 sequences from cloned amplicons revealed that the vast majority (76.97%) differed from the correct sequence by single nucleotide substitutions and/or indels. In total, 34 positions of a 103-bp alignment are affected, and most mutations occur repeatedly in independent PCR amplifications. Several of the induced mutations occur at positions that have previously been detected in studies of ancient hominid sequences, including the Neandertal sequences. Our data imply that PCR-induced mutations are likely to be an intrinsic and general problem of PCR amplifications of ancient templates. Therefore, ancient DNA sequences should be considered with caution, at least as long as the molecular basis for the extract-induced mutations is not understood.