共查询到20条相似文献,搜索用时 0 毫秒
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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. 相似文献
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To test the accuracy of the point methods a simulation was conducted using simulated stomachs. Results from seven participants told to analyse the stomachs by the point and percentage methods differed considerably among subjects and from the control. 相似文献
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Accurate identification of species that are consumed by vertebrate predators is necessary for understanding marine food webs. Morphological methods for identifying prey components after consumption often fail to make accurate identifications of invertebrates because prey morphology becomes damaged during capture, ingestion and digestion. Another disadvantage of morphological methods for prey identification is that they often involve sampling procedures that are disruptive for the predator, such as stomach flushing or lethal collection. We have developed a DNA-based method for identifying species of krill (Crustacea: Malacostraca), an enormously abundant group of invertebrates that are directly consumed by many groups of marine vertebrates. The DNA-based approach allows identification of krill species present in samples of vertebrate stomach contents, vomit, and, more importantly, faeces. Utilizing samples of faeces from vertebrate predators minimizes the impact of dietary studies on the subject animals. We demonstrate our method first on samples of Adelie penguin (Pygoscelis adeliae) stomach contents, where DNA-based species identification can be confirmed by prey morphology. We then apply the method to faeces of Adelie penguins and to faeces of the endangered pygmy blue whale (Balaenoptera musculus brevicauda). In each of these cases, krill species consumed by the predators could be identified from their DNA present in faeces or stomach contents. 相似文献
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Walleye pollock (Theragra chalcogramma) otoliths (n= 2,706) recovered from stomachs, small intestines, and colons of 43 northern fur seals (Callorhinus ursinus) were evaluated for size and wear by location in the digestive tract. Pollock fork length was regressed on otolith length after correction for erosion, and age was estimated from the calculated body size. Age‐1+ pollock otoliths (≥6.3‐mm length) were concentrated in stomachs while age‐0 otoliths (≤6.2‐mm length) were concentrated in colons. Less than 10% of otoliths were found in the small intestines. Pollock age decreased with progression along seal gastrointestinal tracts. Otolith quality increased along gastrointestinal tracts in numbers ≥20, which was typical of age‐0 otoliths recovered from colons. Otolith distribution by age and quality along gastrointestinal tracts suggests that small (≤12 cm) schooling prey are consumed in large volume and passed as a bolus rapidly through the digestive tract before significant erosion of bony remains occurs; while larger prey are eaten in smaller volume and subjected to otolith erosion due to longer retention in the stomach. Our results illustrate the importance of multiple sampling strategies to comprehensively represent prey size in pinniped diet. 相似文献
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Identification and quantitation of species in complex DNA mixtures by real-time polymerase chain reaction 总被引:10,自引:0,他引:10
López-Andreo M Lugo L Garrido-Pertierra A Prieto MI Puyet A 《Analytical biochemistry》2005,339(1):73-82
Six TaqMan real-time polymerase chain reaction (PCR) systems using minor groove binding (MGB) probes have been developed for the detection quantitation of bovine, porcine, lamb, chicken, turkey, and ostrich DNA in complex samples. Species-specific amplification was achieved by combining only two fluorogenic probes and 10 oligonucleotide primers targeting mitochondrial sequences, decreasing the cost of the assay significantly. The limits of detection ranged from 0.03 to 0.80 pg of template DNA. Analysis of experimental mixtures containing two to four different species showed the suitability of the assay for detection of more than 1% of pork, chicken, or turkey and of more than 5% of cattle or lamb. The quantitation accuracy in samples containing 10-100% of beef or pork DNA was close to 90%. The system is complemented with one additional TaqMan MGB detector based on consensus sequence segments of the nuclear 18S ribosomal RNA gene. A method to evaluate the presence of unknown eukaryotic DNA in a mixture, where data derived from the species-specific detection are compared with the experimental values obtained from the general 18S detector, is presented. This method allows the validation of the quantitative measurements, providing an internal control of the total content of PCR-amplifiable DNA in the sample. The system was tested on DNA mixtures containing different shares of up to four different species and on DNA extracted from processed commercial food samples. 相似文献
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DNA identification of non-invasive samples is a potentially useful tool for monitoring small mammal species. Here we describe a novel method for identifying five small mammal species: wood mouse, bank vole, common shrew, pygmy shrew and water shrew. Species-specific real-time polymerase chain reaction primers were designed to amplify fragments of the mitochondrial cytochrome b gene from hair and scat samples. We also amplified nuclear DNA from scats, demonstrating their potential as a source of DNA for population genetic studies. 相似文献
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Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction 总被引:9,自引:0,他引:9
Real-time polymerase chain reaction (PCR) is a highly sensitive method that can be used for the detection and quantification of microbial populations without cultivating them in anaerobic processes and environmental samples. This work was conducted to design primer and probe sets for the detection of methanogens using a real-time PCR with the TaqMan system. Six group-specific methanogenic primer and probe sets were designed. These sets separately detect four orders (Methanococcales, Methanobacteriales, Methanomicrobiales, and Methanosarcinales) along with two families (Methanosarcinaceae and Methanosaetaceae) of the order Methanosarcinales. We also designed the universal primer and probe sets that specifically detect the 16S rDNA of prokaryotes and of the domain Bacteria and Archaea, and which are fully compatible with the TaqMan real-time PCR system. Target-group specificity of each primer and probe set was empirically verified by testing DNA isolated from 28 archaeal cultures and by analyzing potential false results. In general, each primer and probe set was very specific to the target group. The primer and probe sets designed in this study can be used to detect and quantify the order-level (family-level in the case of Methanosarcinales) methanogenic groups in anaerobic biological processes and various environments. 相似文献
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Jonathan Shum 《Analytical biochemistry》2009,388(2):266-3618
Multiplex polymerase chain reaction (PCR), the amplification of multiple targets in a single reaction, presents a new set of challenges that further complicate more traditional PCR setups. These complications include a greater probability for nonspecific amplicon formation and for imbalanced amplification of different targets, each of which can compromise quantification and detection of multiple targets. Despite these difficulties, multiplex PCR is frequently used in applications such as pathogen detection, RNA quantification, mutation analysis, and (recently) next generation DNA sequencing. Here we investigated the utility of primers with one or two thermolabile 4-oxo-1-pentyl phosphotriester modifications in improving multiplex PCR performance. Initial endpoint and real-time analyses revealed a decrease in off-target amplification and a subsequent increase in amplicon yield. Furthermore, the use of modified primers in multiplex setups revealed a greater limit of detection and more uniform amplification of each target as compared with unmodified primers. Overall, the thermolabile modified primers present a novel and exciting avenue for improving multiplex PCR performance. 相似文献
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Joana Romero Carolina Vieira Susana Garrido Margarida Hermida Paulo Catry Graça Faria José Pedro Granadeiro 《Journal of fish biology》2021,99(3):831-843
The Atlantic chub mackerel Scomber colias and the blue jack mackerel Trachurus picturatus are two abundant species in the Macaronesia region which includes the archipelago of Madeira, Portugal. Both are key species in the trophic web, being important prey for several local top predators, such as seabirds and marine mammals. Nonetheless, little is known about their feeding ecology in oceanic environments. In this study, the authors describe the seasonal variation in the diet of S. colias and T. picturatus in the oceanic region of Madeira throughout a year. Visual inspection of stomach contents revealed that S. colias fed on a broader range of prey groups than T. picturatus, but for both species, zooplankton (particularly calanoid copepods) and fish were the most important food items. The diet of S. colias included a higher proportion of fish, namely Atlantic saury Scomberesox saurus and S. colias, than that of T. picturatus, that included mostly the longspine snipefish Macroramphosus scolopax. T. picturatus consumed a higher proportion of decapods and other copepods. Seasonal variation was found in the diet of both species, with zooplanktonic species being more important in colder months (February to April) for S. colias and during warm months (May to October) for T. picturatus. Their diet in other seasons was dominated by fish. Although they consume similar prey, carbon and nitrogen stable isotope analysis of muscle of S. colias and T. picturatus showed little overlap in their diets, and T. picturatus showed higher δ15N and a narrower isotopic niche. 相似文献
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Marshall HD Hart KA Yaskowiak ES Stenson GB McKinnon D Perry EA 《Molecular ecology resources》2010,10(1):181-189
All methods of diet analysis in marine mammals, including hard part analysis (HPA), have biases affecting the accuracy of prey-species identification and frequency in the estimated diet due to differential consumption, digestion and retention. Using PCR amplification of specific prey DNA with species-specific primers, we developed a DNA-based method that complements HPA and provides an alternative means to detect prey from stomach contents of Harp Seals (Pagophilus groenlandicus). The target size that could be reliably amplified was determined using a digestion time-series of Atlantic Cod (Gadus morhua) tissue in simulated seal stomachs. Various target lengths were trialed using general teleost primers; amplicons of approximately 800 bp or less were consistently obtained. Prey species-specific PCR primers for Atlantic Cod, Arctic Cod (Boreogadus saida) and Capelin (Mallotus villosus) were designed and tested with DNA from the stomach contents of 31 Harp Seals. Amplicons were obtained for all three species-specific primer sets. Amplification results compared with HPA revealed: (i) Atlantic Cod hard parts were found in five stomachs where no Atlantic Cod DNA amplified, suggesting that Atlantic Cod may be over-represented in the estimated diet, (ii) amplification of Arctic Cod DNA occurred for 17 stomachs, including all 12 stomachs with, and five stomachs without, Arctic Cod hard parts, and (iii) Capelin DNA amplified for four of five stomachs with Capelin hard parts and for one stomach without Capelin hard parts. We conclude that PCR amplification of specific prey DNA provides a viable means to complement Harp Seal diet analysis by HPA, but suggest that valuable information for quantitative diet analysis rests in a quantitative PCR approach. 相似文献
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随着分子生物学技术的发展, 海洋食品物种鉴定方法由原来的蛋白质水平深入到了DNA水平。目前应用于海洋食品物种鉴定的DNA技术主要是FINS(Forensically informative nucleotide sequencing)、PCR-RFLP和物种特异性PCR标记技术等, 能够实现对新鲜、冰冻、腌制或灌装食品物种进行鉴定, 而对混合样本的鉴定及量化分析是尚待解决的一个问题。基因数据库对物种鉴定的影响也越来越大, 是海洋加工食品物种鉴定可利用的另一种重要信息资源。文章综述了DNA技术在海洋食品物种鉴定中的应用研究进展, 并展望DNA技术在海洋食品检测中的发展趋势。 相似文献
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Morphological identification of many fairy shrimp species is difficult because distinguishing characters are restricted to adults. We developed two multiplex polymerase chain reaction assays that differentiate among three Branchinecta fairy shrimp with distributional overlap in southern California vernal pools. Two of the species are federally listed as threatened. Molecular identification of Branchinecta from cysts allows for species surveys to be conducted during the dry season, expanding the timeframe for population assessment and providing a less intrusive method of sampling sensitive vernal pool habitats. 相似文献
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Applications of real-time polymerase chain reaction for quantification of microorganisms in environmental samples 总被引:11,自引:0,他引:11
Due to the advanced development of fluorogenic chemistry, quantitative real-time polymerase chain reaction (qRT-PCR) has become an emerging technique for the detection and quantification of microorganisms in the environment. Compared with the conventional hybridization- and PCR-based techniques, qRT-PCR not only has better sensitivity and reproducibility, but it is also quicker to perform and has a minimum risk of amplicon carryover contamination. This article reviews the principle of this emerging technique, its detection reagents, target DNAs, quantification procedures, and affecting factors. The applications of qRT-PCR for the quantification of microorganisms in the environment are also summarized. 相似文献
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Tatsuyuki Mimori Ji-ichiro Sasaki Motomi Nakata Eduardo A Gomez Hiroshi Uezato Shigeo Nonaka Yoshihisa Hashiguchi Masato Furuya Hideyuki Saya 《Gene》1998,210(2):487-186
The precise identification and classification of Leishmania species is important for public health surveillance since different species cause different clinical features of the disease. A highly specific polymerase chain reaction (PCR) panel was developed to enable the identification of the five major Leishmania species that cause New World cutaneous leishmaniases. The primers used for this panel were designed to distinguish the polymorphism in sequences of commonly amplified DNA bands of the parasites produced by arbitrarily primed PCR. These polymorphism-specific PCR diagnoses were performed with formalin-fixed biopsy specimens of the leishmanial lesions from four patients in Ecuador and one hamster skin lesion, and these lesions were determined to be caused by Leishmania (Viannia) panamensis, L. (Leishmania) mexicana, and L. (L.) amazonensis. The PCR panel may offer an important and practical approach to the standardized identification of Leishmania species in field examinations. 相似文献