Species identification refined by molecular scatology in a community of sympatric carnivores in Xinjiang,China

Many ecological studies and conservation management plans employ noninvasive scat sampling based on the assumption that species’ scats can be correctly identified in the field. However, in habitats with sympatric similarly sized carnivores, misidentification of scats is frequent and can lead to bias in research results. To address the scat identification dilemma, molecular scatology techniques have been developed to extract DNA from the donor cells present on the outer lining of the scat samples. A total of 100 samples were collected in the winter of 2009 and 2011 in Taxkorgan region of Xinjiang, China. DNA was extracted successfully from 88% of samples and genetic species identification showed that more than half the scats identified in the field as snow leopard(Panthera uncia) actually belonged to fox(Vulpes vulpes). Correlation between scat characteristics and species were investigated, showing that diameter and dry weight of the scat were significantly different between the species. However it was not possible to define a precise range of values for each species because of extensive overlap between the morphological values. This preliminary study confirms that identification of snow leopard feces in the field is misleading. Research that relies upon scat samples to assess distribution or diet of the snow leopard should therefore employ molecular scatology techniques. These methods are financially accessible and employ relatively simple laboratory procedures that can give an indisputable response to species identification from scats.     

PCR检测中国对虾暴发性流行病毒靶基因的克隆和序列分析

An explosive epidemic disease of shrimp had occurred in China and the south Pacific coast. The causative agent is a new baculovirus called hypodermal and hematopoietic necrosis baculovirus (HHNBV) in China, and the pathogen is very much similar to Japanese panaeid rod-shaped DNA virus (PRDV) which caused mass mortality of shrimp. In order to distinguish both HHNBV and PRDV at molecular level, we have sequenced the PCR-targeted DNA fragment of HHNBV. One pair of PCR primers were prepared based on the PRDV DNA. The sequence of the PCR-targeted DNA of HHNBV is 975bp in length, and shares 99.7% homology with the PRDV. The sequence of PCR-targeted DNA of HHNBV-XIA differs from PRDV DNA in deleting three nucleotides (CAT) at 510～512 site. These characteristics of the sequence classified HHNBV and PRDV as strains of different genotypes.     

An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA Extraction

Microbes are ubiquitously distributed in nature, and recent culture-independent studies have highlighted the significance of gut microbiota in human health and disease. Fecal DNA is the primary source for the majority of human gut microbiome studies. However, further improvement is needed to obtain fecal metagenomic DNA with sufficient amount and good quality but low host genomic DNA contamination. In the current study, we demonstrate a quick, robust, unbiased,and cost-effective method for the isolation of high molecular weight(23 kb) metagenomic DNA(260/280 ratio 1.8) with a good yield(55.8 ± 3.8 ng/mg of feces). We also confirm that there is very low human genomic DNA contamination(eubacterial: human genomic DNA marker genes = 2~(27.9):1) in the human feces. The newly-developed method robustly performs for fresh as well as stored fecal samples as demonstrated by 16 S r RNA gene sequencing using 454 FLX+.Moreover, 16 S r RNA gene analysis indicated that compared to other DNA extraction methods tested, the fecal metagenomic DNA isolated with current methodology retains species richnessand does not show microbial diversity biases, which is further confirmed by q PCR with a known quantity of spike-in genomes. Overall, our data highlight a protocol with a balance between quality,amount, user-friendliness, and cost effectiveness for its suitability toward usage for cultureindependent analysis of the human gut microbiome, which provides a robust solution to overcome key issues associated with fecal metagenomic DNA isolation in human gut microbiome studies.     

基因芯片技术检测肾综合征出血热病毒核酸的研究

To establish a rapid, sensitive and specific diagnostic assay for Hantavirus with microarray techniques, specific primers and probes were designed according to the conservative and specific DNA sequence of 76-118 strain and R22 strain. The probes were spotted on glass slides to form microarrays.The Cy3-1abled single stranded DNA fragments prepared by dissymmetical PCR were hybridized with the probes on the glass slides. The microarrays were scanned and analyzed with a scanner. The results showed that the DNA microarray could detect the different typed DNA of HTN and SEO with adequate specificity and sensitivity. The developed DNA microarray and techniques might be a very useful method for diagnosis and prevention, and could be widely applied in specific pathogens detection ofinfectious diseases such as hemorrhagic fever with renal syndrome.     

A hot start alternative for high-fidelity DNA polymerase amplification mediated by quantum dots

Quantum dots （QDs） are of great interest due to their unique chemical and physical properties. Recently, a hot start （HS） polymerase chain reaction （PCR） amplification performance based on QDs with a high-fidelity Pfu DNA polymerase has been reported. However, whether QDs can trigger HS effects with other high-fidelity or conventional DNA polymerases is yet to be understood. In the present study, we studied the QD-triggered HS effects with four high-fidelity and three conventional DNA polymerases, and the HS effect comparisons among them were also made. It was found that QDs could trigger a distinct HS PCR amplification performance with all the four tested high,fidelity DNA polymerases, and specific target DNA could be well amplified even if the PCR mixture was preincubated for 2 h at 50℃. On the contrary, the HS effects were not prominent with all the three conventional Taq DNA polymerases. Specifically, the fidelity of Pfu is not sacrificed in the presence of QDs, even after a 1 h pre-incu- bation at 50℃ before PCR. Furthermore, the electrophoresis results preliminarily demonstrated that QDs prefer to adsorb high-fidelity polymerases rather than conventional ones, which might result in the QD-triggered HS effects on PCR performance by using high-fidelity DNA poly- merases.     

Transmission identification of Escherichia coli aerosol in chicken houses to their environments using ERIC-PCR

In order to study E. coli aerosol spreading from chicken houses to their surrounding air, air samples, including indoor and outdoor air (upwind 10 and 50 m as well as downwind 10, 50, 100, 200 and 400 m away) of 5 chicken houses were collected using six-stage Andersen microbial samplers and Reuter-Centrifugal samplers (RCS). E. coli concentrations (CFU/m3 air) collected from different sampling sites were calculated. E. coli strains from chicken feces samples were also isolated. Furthermore, the enterobacterial repetitive intergenic consensus (ERIC)-PCR method was applied to amplify the isolated E. coli strain DNA samples. Through the genetic similarity analyses of the E. coli obtained from different sampling sites, the spreading of bioaerosol from animal houses to the ambient air was characterized. The results showed that the isolated E. coli concentrations in indoor air (9―63 CFU/m3) in 5 chicken houses were higher than those in upwind and downwind air, but there were no significant differences between the indoor and downwind sites 10 m away from all the 5 houses (P>0.05). The phylogenetic tree indicated that a part of the E. coli (34.1%) isolated from indoor air had 100% similarity with those isolated from feces, and that most of E. coli isolated (54.5%) from downwind at 10, 50, 100 or even 200 m had 100% similarity with those isolated from indoor air or feces too. But those isolated from upwind air had a lower similarity (73%―92%) with corresponding strains isolated from indoor air or feces. Our results suggested that some strains isolated from downwind air and indoor air originated in the chicken feces, but most of isolates obtained from upwind air samples did not come from the chicken feces or indoor air. Effective hygienic measures should be taken in animal farms to prevent or minimize downwind spreading of microorganism aerosol.     

A meta-analysis of the effects of geolocator application on birds

An increasing trend in use of tracking devices such as geolocators is based on the assumption that the information gathered from such devices provides reliable information about the migratory behavior of free-living birds. This underlying as- sumption is rarely tested, as evidenced by the absence in many studies of controls, in particular treated controls, and so far never with a reasonable statistical power. Published studies have shown reduced survival prospects or delayed breeding in some species, suggesting that there may be reason to doubt that tracking devices provide unbiased information. Therefore, we conducted a meta-analysis of studies applying geolocators to wild birds to determine whether geolocators affected fitness components. Ge- olocators had an overall negative effect on fitness components, in particular survival, and ecological variables. Effect size was larger for aerial foragers than for other species. Moreover the leg band attachment method was more detrimental for birds than the leg-loop backpack harness. A meta-regression model of effect size showed independent negative effects of geolocators on ae- rial foragers, smaller species, species with smaller migration distances and in studies where geolocators were attached with a ring. These results suggest that geolocator studies should be interpreted with caution, but also raise questions whether it is ethically defensible to use geolocators on aerial foragers or small species without carrying out robust pilot studies.     

Rapid and low-level toxic PCR-based method for routine identification of Flavobacterium psychrophilum.

We describe a rapid, low-toxicity and simple method for the detection of the bacterial fish pathogen Flavobacterium psychrophilum. The method, based on the polymerase chain reaction (PCR), combined the electrophoresis of PCR products in a vertical agarose gel and a modified methylene blue stain. DNA was amplified directly either from bacterial suspensions or from tissues experimentally infected with F. psychrophilum, using different non-toxic commercial DNA extraction kits. The protocol allowed to detect 15 to 150 cells of the pathogen in bacterial suspension, without prior DNA extraction, and 7500 to 75,000 cells in seeded spleen tissue and ovarian fluid using Dynabeads DNA DIRECT extraction system. This method, which has the advantage of not using hazardous products, is proposed as a fast tool for routine identification of F. psychrophilum.     

Quantitative PCR detection of Batrachochytrium dendrobatidis DNA from sediments and water

The fungal pathogen Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis, a disease implicated in amphibian declines on 5 continents. Polymerase chain reaction (PCR) primer sets exist with which amphibians can be tested for this disease, and advances in sampling techniques allow non-invasive testing of animals. We developed filtering and PCR based quantitative methods by modifying existing PCR assays to detect Bd DNA in water and sediments, without the need for testing amphibians; we tested the methods at 4 field sites. The SYBR based assay using Boyle primers (SYBR/Boyle assay) and the Taqman based assay using Wood primers performed similarly with samples generated in the laboratory (Bd spiked filters), but the SYBR/Boyle assay detected Bd DNA in more field samples. We detected Bd DNA in water from 3 of 4 sites tested, including one pond historically negative for chytridiomycosis. Zoospore equivalents in sampled water ranged from 19 to 454 1(-1) (nominal detection limit is 10 DNA copies, or about 0.06 zoospore). We did not detect DNA of Bd from sediments collected at any sites. Our filtering and amplification methods provide a new tool to investigate critical aspects of Bd in the environment.     

Molecular detection of Puccinia horiana in Chrysanthemum x morifolium through conventional and real-time PCR

Puccinia horiana Henn. is a quarantine organism and one of the most important fungal pathogens of Chrysanthemum x morifolium cultivars grown for cut flower or potted plant production (florist's chrysanthemum) in several regions of the world. Highly specific primer pairs were identified for conventional, nested, and real-time PCR detection of P. horiana based on the specific and sensitive PCR amplification of selected regions in the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA (rDNA). Using these different PCR versions, 10 pg, 10 fg, and 5 fg genomic DNA could be detected, respectively. When using cloned target DNA as template, the detection limits were 5000, 50, and 5 target copies, respectively. These detection limits were not affected by a background of chrysanthemum plant DNA. The DNA extraction method was optimized to maximize the recoverability of the pathogen from infected plant tissue. A CTAB extraction protocol or a selection of commercial DNA extraction methods allowed the use of 10 ng total (plant+pathogen) DNA without interference of PCR inhibitors. Due to the specificity of the primers, SYBR Green I technology enabled reliable real time PCR signal detection. However, an efficient TaqMan probe is available. The lowest proportion of infected plant material that could still be detected when mixed with healthy plant material was 0.001%. The real-time PCR assay could detect as few as eight pure P. horiana basidiospores, demonstrating the potential of the technique for aerial detection of the pathogen. The amount of P. horiana DNA in plant tissue was determined at various time points after basidiospore inoculation. Using the real-time PCR protocol, it was possible to detect the pathogen immediately after the inoculation period, even though the accumulation of pathogen DNA was most pronounced near the end of the latent period. The detection system proved to be accurate and sensitive and could help not only in pathogen diagnosis but also in pathogen monitoring and disease forecasting systems.     

Japanese macaque microsatellite PCR primers for paternity testing

Capture and blood sampling in wild primate populations are difficult. For this reason, we need to use DNA extracted from the hair or feces of target animals. The polymerase chain reaction (PCR) method, which amplifies small volumes of DNA, provides an ideal means for studying DNA variations in wild populations. Three sets of PCR primers which amplify highly polymorphic (GT/AC)_n dinucleotide repetitive regions were synthesized from DNA sequences of Japanese macaques (Macaca fuscata). One of the primer pairs detected at least seven alleles in one captive Japanese macaque group. Also, the fathers of four offspring whose mothers had died in a captive group of Japanese macaques were identified. In such cases, the father cannot be determined by the previous DNA fingerprinting method based on the polymorphism of minisatellite DNA. These primers were further tested with some species of the Cercopithecidae, e.g. grivet monkeys (Cercopithecus aethiops tantalus) and hamadryas baboons (Papio hamadryas). The results obtained suggest that these primers can detect stably inherited polymorphic regions in each species.     

Real-time PCR for the detection of Cryptosporidium parvum.

Real time, TaqMan PCR assays were developed for the Cp11 and 18S rRNA genes of the protozoan parasite Cryptosporidium parvum. The TaqMan probes were specific for the genus Cryptosporidium, but could not hybridize exclusively with human-infectious C. parvum species and genotypes. In conjunction with development of the TaqMan assays, two commercial kits, the Mo Bio UltraClean Soil DNA kit, and the Qiagen QIAamp DNA Stool kit, were evaluated for DNA extraction from calf diarrhea and manure, and potassium dichromate and formalin preserved human feces. Real-time quantitation was achieved with the diarrhea samples, but nested PCR was necessary to detect C. parvum DNA in manure and human feces. Ileal tissues were obtained from calves at 3, 7, and 14 days post-infection, and DNA extracted and assayed. Nested PCR detected C. parvum DNA in the 7-day post-infection sample, but neither of the other time point samples were positive. These results indicate that real-time quantitation of C. parvum DNA, extracted using the commercial kits, is feasible on diarrheic feces, with large numbers of oocysts and small concentrations of PCR inhibitor(s). For samples with few oocysts and high concentrations of PCR inhibitor(s), such as manure, nested PCR is necessary for detection.     

Rapid detection of human fecal Eubacterium species and related genera by nested PCR method

PCR procedures based on 16S rDNA gene sequence specific for seven Eubacterium spp. and Eggerthella lenta that predominate in the human intestinal tract were developed, and used for direct detection of these species in seven human feces samples. Three species of Eggerthella lenta, Eubacterium rectale, and Eubacterium eligens were detected from seven fecal samples. Eubacterium biforme was detected from six samples. It was reported that E. rectale, E. eligens, and E. biforme were difficult to detect by traditional culture method, but the nested PCR method is available for the detection of these species. This result shows that the nested PCR method utilizing a universal primer pair, followed by amplification with species-specific primers, would allow rapid detection of Eubacterium species in human feces.     

Real-time PCR detection of Biscogniauxia mediterranea in symptomless oak tissue

AIMS: Real-time PCR, based on TaqMan chemistry, was used to detect Biscogniauxia mediterranea, a fungal pathogen that after a long endophytic phase may cause charcoal disease in oak trees. METHODS AND RESULTS: Specific primers and probe were designed and tested on axenic cultures of B. mediterranea and other fungi commonly colonizing oaks. Twig samples were collected in Tuscany from apparently healthy oaks (Quercus cerris, Quercus ilex and Quercus pubescens) growing near trees infected with the fungus. Twigs were divided into two groups: one for isolation in agar plates, and one for real-time PCR after DNA extraction. The detection limit of the assay was 0.01 pg/DNA, whereas the amounts of fungal DNA detected in asymptomatic tissue were >0.5 pg microg(-1) total DNA extracted. In the apparently healthy twigs the frequency of isolation found on agar was 25.0%, much lower than that with real-time PCR (96.4%). CONCLUSIONS: Real-time PCR is a sensitive and fast technique able to specifically detect and quantify the DNA of B. mediterranea in oak tissue. SIGNIFICANCE AND IMPACT OF THE STUDY: This diagnostic method is a precise tool to localize fungi in symptomless plant tissues and promises to advance our understanding of fungal infection during their latent phase.     

Use of PCR for direct detection of Campylobacter species in bovine feces

This study reports on the use of PCR to directly detect and distinguish Campylobacter species in bovine feces without enrichment. Inhibitors present in feces are a major obstacle to using PCR to detect microorganisms. The QIAamp DNA stool minikit was found to be an efficacious extraction method, as determined by the positive amplification of internal control DNA added to bovine feces before extraction. With nested or seminested multiplex PCR, Campylobacter coli, C. fetus, C. hyointestinalis, and C. jejuni were detected in all fecal samples inoculated at approximately 10(4) CFU g(-1), and 50 to 83% of the samples inoculated at approximately 10(3) CFU g(-1) were positive. At approximately 10(2) CFU g(-1), C. fetus, C. hyointestinalis, and C. jejuni (17 to 50% of the samples) but not C. coli were detected by PCR. From uninoculated bovine feces, a total of 198 arbitrarily selected isolates of Campylobacter were recovered on four commonly used isolation media incubated at three temperatures. The most frequently isolated taxa were C. jejuni (152 isolates) and C. lanienae (42 isolates), but isolates of C. fetus subsp. fetus, Arcobacter butzleri, and A. skirrowii also were recovered (相似文献   

Detection of kinetoplast DNA of Trypanosoma cruzi from dried feces of triatomine bugs by PCR.

It is important to clarify the distribution of infected triatomine bugs in the endemic area of Chagas' disease for proper control. In the present study, we tried to detect T. cruzi kinetoplast DNA by PCR from dried triatomine feces collected from the house wall of an endemic area to assess the distribution of infected bugs more easily. The primers (P35/P36) were chosen to amplify the conserved region within the minirepeats of T. cruzi kinetoplast minicircle DNA. The kinetoplast DNA of T. cruzi could be actually detected in the dried feces collected from the wall of a brick-built house in Santa Cruz, Bolivia. Next, we examined the stability of T. cruzi kinetoplast DNA in the feces exposed to artificial environments. T. cruzi DNA was also detected by PCR in the feces left for 26 weeks at 25 degrees C and in those left for 4 weeks at 40 degrees C. The present study indicates that examination of dried feces on the wall can be an effective tool for surveillance of the natural infection of triatomine bugs that live in houses.     

Pre-PCR processing

Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.