Pyrosequencing检测口腔与胃中的幽门螺杆菌16SrDNAV1区基因序列

目的通过Pyrosequencing检测口腔与胃幽门螺杆菌16SrDNAV1区序列基因片段分析,以进一步验证口—口传播的的假设。方法选择18例患有慢性中度牙周炎且胃镜活检Hp感染阳性患者及其中10例患者的家属。提取患者胃、牙菌斑和含漱液共74例样本中的DNA,PCR扩增阳性后pyrosequecing检测16SrDNAV1区序列基因片段。结果患者胃和口腔Hp及其家属口腔Hp的序列相比较,有0～1个碱基不同。结论口腔中的Hp与胃内Hp16SrDNAV1区基因型比较95.8%～100%的同源性,口腔可能为Hp的聚集地。口腔Hp可能与胃Hp感染的复发或再感染有关。

Pyrosequencing检测CYP2C9^＊3基因多态性方法的建立及其可靠性研究

目的:建立基于焦磷酸测序技术(Pyrose-quencing)的高通量的CYP2C9*3突变检测方法。方法:应用带有生物素标记的扩增引物,经PCR扩增及Beads分离,制备该突变位点焦磷酸测序单链模板,并在PyroMark ID焦磷酸测序仪上进行焦磷酸测序,以经典ABI测序法测序结果作为标准对照,观察批量分析的可靠性并批量检测220例人DNA标本。结果:建立了基于焦磷酸测序技术的CYP2C9*3突变位点检测平台,实现了DNA标本的高通量检测。能一次获得96份DNA的CYP2C9*3突变位点检测结果。经重复性检测和标准的ABI测序可靠性检测,CYP2C9*3突变位点的突变检出率及重复率均达100%。结论:本方法可准确、高通量、快速检测CYP2C9*3突变,特别适宜该SNP位点批量检测需要。

采用Pyrosequencing和Pooling技术对SNP位点多态性分析

目的建立基于pyrosequencing和Pooling技术进行SNP位点的法医学多态性分析技术。方法对50名无关个体样本建立一适合pyrosequencing检测的组池;采用PyroMark Assay Design 2.0软件进行SNP位点等位基因定量分析的引物设计;对组池样本PCR产物进行焦磷酸测序检测。结果检测的3个SNP位点多态性良好,其中位点rs220028与以往人群调查后频率数据无显著差异。结论采用pyrosequencing和Pooling技术对SNP位点进行多态性分析,适合于位点的初筛及大规模群体调查。该技术准确可靠,方便快捷。

Comparative analysis of dideoxy sequencing,the KRAS StripAssay and pyrosequencing for detection of KRAS mutation

AIM:To compare the differences between dideoxy sequencing/KRAS StripAssay/pyrosequencing for detection of KRAS mutation in Chinese colorectal cancer (CRC) patients.METHODS:Formalin-f ixed, paraff in-embedded (FFPE) samples with tumor cells ≥ 50% were collected from 100 Chinese CRC patients at Beijing Cancer Hospital. After the extraction of genome DNA from FFPE samples, fragments contained codons 12 and 13 of KRAS exon 2 were amplified by polymerase chain reaction and analyzed by dideoxy sequencing, the KRAS Strip Assay and pyrosequencing. In addition, the sensitivities of the 3 methods were compared on serial dilutions (contents of mutant DNA: 100%,50%,20%, 5%,10%, 5%,1%,0%) of A549 cell line DNA (carrying the codon 12 Gly>Ser mutation) into wild-type DNA (human normal intestinal mucosa). The results of dideoxy sequencing,the KRAS StripAssay and pyrosequencing were analyzed by Chromas Software, Collector forKRAS Strip Assay and the pyrosequencing PyroMarkTM Q24 system, respectively.RESULTS: Among 100 patients, KRAS mutations were identif ied in 34%, 37% and 37% of patients by dideoxy sequencing, the KRAS StripAssay and pyrosequencing, respectively. The sensitivity was highest with the KRAS Strip Assay (1%), followed by pyrosequencing (5%), and dideoxy sequencing was lowest (15%). Six different mutation types were found in this study with 3 main mutations Gly12 Asp (GGT>GAT), Gly12 Val (GGT>GTT) and Gly13 Asp (GGC>GAC). Thirty-three patients were identifi ed to have KRAS mutations by the 3 methods, and a total of 8 patients had conflicting results between 3 methods: 4 mutations not detected by dideoxy sequencing and the KRAS StripAssay were identified by pyrosequencing; 3 mutations not detected by dideoxy sequencing and pyrosequencing were identif ied by the KRAS StripAssay; and 1 mutation not detected by pyrosequencing was conf irmed by dideoxy sequencing and the KRAS StripAssay. Among these discordant results, the results identif ied by dideoxy sequencing were consistent either with the KRAS StripAssay or with pyrosequencing, which indicated that the accuracy of dideoxy sequencing was high. CONCLUSION: Taking a worldwide view of reports and our results,dideoxy sequencing remains the most popular method because of its low cost and high accuracy.

Consuming fermented distillers＇ dried grains with solubles (DDGS) feed reveals a shift in the faecal microbiota of growing and fattening pigs using 454 pyrosequencing

The objective of this study was to investigate pig fed by Bacillus coagulans-fermented distillers＇ dried grains with solubles （DDGS） on the faecal microbial composition and diversity using 454 pyrosequencing. Healthy crossbred （Durocx Yorkshirex Landrace） growing and fattening pigs （n=48）, with an average initial body weight of 65 kg, were divided into two groups （24 replicates per group; four pens per group; six pigs per pen）, and given either DDGS feed as the control, or B. coagulans-fermented DDGS feed as the treatment. Faecal samples were collected on day 0, 7, 14, 21, and 28. DNA was extracted, and the V3-V6 region of the 16S rRNA gene was amplified. The fermented DDGS feed affected the relative abundance of bacteria populations at the phylum, genus, and species levels. At the genus level, the consumption of fermented DDGS feed led to higher relative abundances of faecal Prevotella, Lactobacillus, Clostridium, Bifidobacterium, Roseburia, and Bacillus, and lower relative abundances of faecal Escherichia, Ruminococcus, Dialister, unclassified Lachnospiraceae, unclassified Ruminococcaceae, and unclassified Enterobacteriaceae than in the control. At the species level, the consumption of fermented DDGS feed led to higher relative abundances of faecal Prevotella sp., Lactobacillus johnsonii, Lactobacillus fermentum, Lactobacillus mucosae, Lactobacillus reuteri, Clostridium butyricum, Bifidobacterium sp., and Roseburia sp., and lower relative abundances of faecal Prevotella copri, Escherichia coil, Ruminococcus gnavus, Ruminococcus flavefaciens, and Dialister sp. than in the control. Principal coordinates analysis indicated a distinct separation in the faecal microbial communities of pigs that were fed the fermented and unfermented DDGS feed. Fermented DDGS feed significantly increased the average daily gain （ADG） of pigs, and significantly decreased the average daily feed intake （ADFI） of feed and feed/gain （F/G）. Thus, our results demonstrate a beneficial shift in the faecal microbiota of pigs consuming fermented DDGS feed, with potential applications in livestock production.

Comparison of amplicon-sequencing, pyrosequencing and real-time PCR for detection of YMDD mutants in patients with chronic hepatitis B

AIM: To compare the sequencing of PCR products, pyro- sequencing, and real-time PCR for detection of Tyrosine- methionine-aspartate-aspartate (YMDD) mutants in patients with chronic hepatitis B. METHODS: Mixtures of plasmids and serum samples from 69 chronic hepatitis B patients treated with lamivu- dine were tested for YMDD mutations by sequencing of PCR products, pyrosequencing, and real-time PCR, re- spectively. Time required and reagent costs of the three assays were evaluated. RESULTS: Real-time PCR detected 100%, 50%, 10%, 1% and 0.1% of YVDD plasmid in mixtures with 106 copies/mL of YMDD plasmid, whereas sequencing and pyrosequencing only detected 100% and 50% of YVDD plasmid in aliquots of the corresponding mixtures. Com- pletely concordant results were obtained from 60 (87%) out of the 69 clinical serum samples by the three assays. Mutants were detected by real-time PCR in less than 20% of the total virus population, but no mutant was de- tected by sequencing and pyrosequencing. In addition, real-time PCR required less time and was more cost-ef- fective than the other two assays. However, throughput of pyrosequencing was the highest. CONCLUSION: Among the three assays compared, real-time PCR is the most sensitive, cost-effective, and time saving for monitoring YMDD mutants in patients with chronic hepatitis B on lamivudine therapy.

Bacterial and archaeal community structure of pan-Arctic Ocean sediments revealed by pyrosequencing

This study was to investigate bacterial and archaeal community structure of pan-Arctic Ocean sediments by pyrosequencing. In total, investigation of three marine sediments revealed 15 002 bacterial and 4 362 archaeal operational taxonomic units （OTUs） at the 97% similarity level. Analysis of community structure indicated that these three samples had high bacterial and archaeal diversity. The most relatively abundant bacterial group in Samples CC 1 and R05 was Proteobacteria, while Firmicutes was dominant in Sample BL03. Thaumarchaeota was the most relatively abundant archaeal phylum in Samples CC1 and R05, and the relative abundance of Thaumarchaeota was almost as high as that of Euryarchaeota in Sample BL03. These two phyla accounted for nearly 100% of the archaeal OTUs. 6-Proteobacteria and y-Proteobacteria were the two most relatively abundant classes at Proteobacterial class level, and their relative abundance was more than 60% in Samples CC1 and R05. There were also differences in the top 10 relatively abundant bacterial and archaeal OTUs among the three samples at the 97% similarity, and only 12 core bacterial OTUs were detected. Overall, this study indicated that there were distinct microbial communities and many unique OTUs in these three samples.

Microbial community structure of Arctic seawater as revealed by pyrosequencing

This study aimed to determine the microbial community structure of seawater in（ICE-1） and out（FUBIAO） of the pack ice zone in the Arctic region.Approximate 10 L seawater was filtrated by 0.2 μm Whatman nuclepore filters and the environmental genomic DNA was extracted.We conducted a detailed census of microbial communities by pyrosequencing.Analysis of the microbial community structures indicated that these two samples had high bacterial,archaeal and eukaryotic diversity.Proteobacteria and Bacteroidetes were the two dominant members of the bacterioplankton community in both samples,and their relative abundance were 51.29% and 35.39%,72.95%and 23.21%,respectively.Euryarchaeota was the most abundant archaeal phylum,and the relative abundance was nearly up to 100% in FUBIAO and 60% in ICE-1.As for the eukaryotes,no_rank_Eukaryota,Arthropoda and no_rank_Metazoa were the most abundant groups in Sample FUBIAO,accounting for 85.29% of the total reads.The relative abundance of the most abundant phylum in Sample ICE-1,no_rank_Eukaryota and no_rank_Metazoa,was up to 90.69% of the total reads.Alphaproteobacteria,Flavobacteria and Gammaproteobacteria were the top three abundant classes in the two samples at the bacterial class level.There were also differences in the top ten abundant bacterial,archaeal and eukaryotic OTUs at the level of 97% similarity between the two samples.

Asymmetric PCR method in generation of HBV ssDNA for pyrosequencing

Objective To explore the optimal primer ratio and concentration of asymmetric polymerase chain reaction (A-PCR) in producing hepatitis B virus (HBV) single-stranded DNA (ssDNA) for pyrosequencing. Methods A-PCR was carried out to generate HBV ssDNA with forward to reverse primers of different ratios (50∶1, 100∶1) and concentrations (13.0 pmol/25μL and 0.14 pmol/25μL, 19.5 pmol/25μL and 0.21 pmol/25μL), and the product yield and quality were compared respectively. Results The forward to reverse primer ratio of 50∶1 provided better yield and concentration of 19.5 pmol/25μL and 0.21 pmol//25μL generated a clearer band. Conclusion A simple and feasible method to produce HBV ssDNA for pyrosequencing in batch is established.

单管PCR-Pyrosequencing快速检测华法林代谢酶基因多态性方法的建立

文章旨在建立一种单管、快速及高通量的华法林药物代谢酶相关基因多态性的检测方法。通过抽取人外周血DNA,应用带有生物素标记的扩增引物,经PCR扩增并制备焦磷酸测序单链模板,于PyroMark ID焦磷酸测序仪上进行焦磷酸测序,以Sanger测序法测序结果为对照,观察分析的准确性。结果显示,华法林药物代谢酶的3个相关基因多态性(CYP2C9*2、CYP2C9*3、VKORC1(-1693))于单管中可被同时检测,一次可获得96份DNA的华法林药物代谢相关多态性位点检测结果。经与Sanger测序方法比较,符合率为100%。结果表明本方法可准确、高通量、快速检测华法林药物代谢酶相关基因多态性,与单管检测一个位点的焦磷酸测序方法相比,能有效降低检测成本,节省检测时间。该方法在个性化医疗上有较大的推广应用价值,也可以将该平台运用于其他疾病相关基因多态性检测。

PCR—Pyrosequencing快速检测药物代谢酶基因CYP2C19多态性方法的建立及应用

目的旨在建立一种快速及高通量的CYP2C19药物代谢酶相关基因多态性的检测方法，为个体化治疗提供用药参考。方法抽取96例人外周血DNA，应用带有生物素标记的扩增引物，经PCR扩增并制备焦磷酸测序单链模板，于PyroMarkID焦磷酸测序仪上进行焦磷酸测序，且以Sanger测序法测序结果为对照，观察分析准确性。结果运用焦磷酸测序可以成功检测CYP2C19相关基因型；单个sNP住点检测经与Sanger测序方法比较，符合率为100％。同时，通过设计焦磷酸测序引物，两个PCR产物可合并于一管中进行焦磷酸测序，与单管检测一个位点的焦磷酸测序方法相比，能有效降低检测成本，节省检测时间。结论该方法可准确、高通量、快速检测药物代谢酶相关基因多态性，该方法在个性化医疗上有较大的推广应用价值，也可以将该平台运用于其他疾病相关基因多态性检测。

Characterization of root-associated bacterial community structures in soybean and corn using locked nucleic acid(LNA) oligonucleotide-PCR clamping and 454 pyrosequencing

supported in part by grants from the Strategic Priority Research Program of Chinese Academy of Sciences （XDB15010103）;the National Natural Science Foundation of China （41201247）

Pyrosequencing-based assessment of bacterial community structure in mine soils affected by mining subsidence

Based on the 454 pyrosequencing approach, this research evaluated the influence of coal mining subsi- dence on soil bacterial diversity and community structure in Chinese mining area. In order to characterize the bacterial community comparatively, this study selected a field experiment site with coal-excavated subsidence soils and an adjacent site with non-disturbed agricultural soils, respectively. The dataset com- prises 24512 sequences that are affiliated to the 7 phylogenetic groups： proteobacteria, actinobacteria, bacteroidetes, gemmatimonadetes, chlorofiexi, nitrospirae and unclassified phylum. Proteobacteria is the largest bacterial phylum in all samples, with a marked shift of the proportions of alpha-, beta-, and gammaproteobacteria. The results show that undisturbed soils are relatively more diverse and rich than subsided soils, and differences in abundances of dominant taxonomic groups between the two soil groups are visible. Compared with the control, soil nutrient contents decline achieves significant level in subsided soils. Correlational analysis showed bacterial diversity indices have significantly positive corre- lation with soil organic matter, total N, total P, and available K. but in negative relation with soil salinity. Ground subsidence noticeably affects the diversity and composition of soil microbial community. Degen- eration of soil fertility and soil salinization inhibits the sole-carbon-source metabolic ability of microbial community, leading to the simplification of advantage species and uneven distribution of microbial spe- cies. This work demonstrates the great potential of pyrosequencing technique in revealing microbial diversity and presents background information of microbial communities of mine subsidence land.

Profile of candidate microsatellite markers in Sebastiscus marmoratus using 454 pyrosequencing

Sebastiscus marmoratus is an important sedentary ovoviparous fish distributed in near-shore coastal waters from the coast of China to Japan. Candidate S. marmoratus microsatellite markers were developed in the present study using 454 pyrosequencing, and the marker profile was analyzed. A total of 2 000 000 raw sequence reads were assembled to reduce redundancy. Among them, 1 043 dinucleotide, 925 trinucleotide, 692 tetranucleotide, and 315 pentanucleotide repeats were detected. AC repeats were the most frequent motifs among the dinucleotide repeats, and AAT was the most abundant among the trinucleotide repeats. AAAT, ATAG, and ATCC were the three most common tetranucleotide motifs, and AAGAT and AATAT were the most dominant pentanucleotide motifs. The greatest numbers of loci and potentially amplifiable loci were found in dinucleotide repeats, whereas trinucleotide repeats had the fewest. In summary, a wide range of candidate microsatellite markers were identified in the present study using a rapid and efficient 454 pyrosequencing approach.

Fecal Microbiota Succession of Piglets from Birth to Post-weaning by 454 Pyrosequencing Analysis

The objective of this study was to describe the fecal microbiota succession of piglets from birth to post-weaning by pyrosequencing. The dominant bacterial phyla were Bacteroidetes, Firmicutes, Proteobacteria, and Fusobacteria, although the composition of the fecal microbiota changed as the piglets grew. Bacteroides, Parabacteroides, Prevotella, Lactobacillus, unclassified Lachnospiraceae, Ruminococcus, Oscillospira, Phascolarctobacterium, and Desulfovibrio were the core genera in the feces of piglets and existed from birth to post-weaning. Bacteroides was the most abundant at birth; however, the proportion of Bacteroides decreased as the piglets aged, while the proportion of Prevotella increased until it became the most abundant genus at the post-weaning stage. Principal coordinate analysis indicated that the microbial communities of piglet fecal samples from birth to post-weaning could be separated into four groups according to the similarity of community composition. These corresponded to time points on day 0 (birth); day 7; days 14, 21, and 28 (pre-weaning); and day 35 (post-weaning). This study suggests that the development of the intestinal microbiota in the piglet is a gradual and sequential process. This methodology establishes a basis for broader studies to investigate the microbiota of the piglet intestinal tract. © 2017, Tianjin University and Springer-Verlag Berlin Heidelberg.

Establishment of Pyrosequencing Technology for Detecting Viral Hemorrhagic Septicemia Virus (VHSV)

[Objective] The paper was to establish pyrosequencing methods for detecting viral hemorrhagic septicemia virus （VHSV）. [ Method ] One pair of PCR primers and one pyrosequencing primer of VHSV were designed. The pyrosequencing reaction system and conditions were optimized and the pyrosequencing method for detecting VHSV was established. [ Result] This method was only able to specifically detect the objective viruses in the eight fish viruses, and the method had the advantage of high sensitivity. The minimum detectable limit of nucleic acid was 82 copies/μL. The method was verified by detecting VHSV in 1 924 batches of samples collected from domestic and imported fishes. The detection results were consistent with that of traditional RT-PCR, and the specificity and sensitivity of the method could meet the detection requirement for aquatic animal diseases. [ Conclusion] The study provides a new detection method for monitoring and prevention and control of aquatic animal virus diseases.

High-throughput calculations combining machine learning to investigate the corrosion properties of binary Mg alloys

Magnesium(Mg)alloys have shown great prospects as both structural and biomedical materials,while poor corrosion resistance limits their further application.In this work,to avoid the time-consuming and laborious experiment trial,a high-throughput computational strategy based on first-principles calculations is designed for screening corrosion-resistant binary Mg alloy with intermetallics,from both the thermodynamic and kinetic perspectives.The stable binary Mg intermetallics with low equilibrium potential difference with respect to the Mg matrix are firstly identified.Then,the hydrogen adsorption energies on the surfaces of these Mg intermetallics are calculated,and the corrosion exchange current density is further calculated by a hydrogen evolution reaction(HER)kinetic model.Several intermetallics,e.g.Y_(3)Mg,Y_(2)Mg and La_(5)Mg,are identified to be promising intermetallics which might effectively hinder the cathodic HER.Furthermore,machine learning(ML)models are developed to predict Mg intermetallics with proper hydrogen adsorption energy employing work function(W_(f))and weighted first ionization energy(WFIE).The generalization of the ML models is tested on five new binary Mg intermetallics with the average root mean square error(RMSE)of 0.11 eV.This study not only predicts some promising binary Mg intermetallics which may suppress the galvanic corrosion,but also provides a high-throughput screening strategy and ML models for the design of corrosion-resistant alloy,which can be extended to ternary Mg alloys or other alloy systems.

Utilization of Pyrosequencing to Monitor the Microbiome Dynamics of Probiotic Treated Poultry (<i>Gallus gallus domesticus</i>) during Downstream Poultry Processing

Antibiotic growth promoters that have been historically employed to control pathogens and increase the rate of animal development for human consumption are currently banned in many countries. Probiotics have been proposed as an alternative to control pathogenic bacteria. Traditional culture methods typically used to monitor probiotic effects on pathogens possess significant limitations such as a lack in sensitivity to detect fastidious and non-culturable bacteria, and are both time consuming and costly. Here, we tested next generation pyrosequencing technology as a streamline and economical method to monitor the effects of a probiotic on microbial communities in juvenile poultry (Gallus gallus domesticus) after exposure to several microbiological challenges and litter conditions. Seven days and repeated again at 39 days following hatching, chicks were challenged with either Salmonella enterica serovar Enteritidis, Campylobacter jejuni, or no bacteria in the presence of, or without a probiotic (i.e., Bacillus subtilis) added to the feed. Three days following each of two challenges (i.e., days 10 and 42, respectively) the microbiome distributions of the poultry caecum were characterized based on 16S rDNA analysis. Generated PCR products were analyzed by automated identification of the samples after pooling, multiplexing and sequencing. A bioinformatics pipeline was then employed to identify microbial distributions at the phylum and genus level for the treatments. In conclusion, our results demonstrated that pyrosequencing technology is a rapid, efficient and cost-effective method to monitor the effects of probiotics on the microbiome of poultry propagated in an agricultural setting.

Evaluation of Real-Time 16S rDNA PCR and Pyrosequencing for Routine Identification of Bacteria in Joint Fluid and Tissue Specimens

16S rDNA PCR and sequencing are powerful tools for bacterial detection and identification, although their routine use is not currently widespread in the field of clinical microbiology. The availability of pyrosequencing now makes 16S rDNA assays more accessible to routine diagnostic laboratories, but this approach has had limited evaluation in general diagnostic practice. In this study we evaluated a real-time 16S rDNA PCR and pyrosequencing assay for use in a routine microbiology laboratory, by retrospectively testing joint fluid and joint tissue specimens received for conventional culture. We found that use of the real-time 16S rDNA assay was clinically valuable in this specimen type because it enabled us to identify a small number of culture-negative infections. Although faster and less labour-intensive, we found that the utility of pyrosequencing for pathogen identification is still hampered by shorter read lengths compared to conventional (Sanger) sequencing. Combining results from both molecular and conventional culture methods, bacteria were only detected in 11.8% specimens in this study. However, the detection rate was increased to 18.6% if specimens were only included from patients with a documented clinical suspicion of infection. In conclusion, while pyrosequencing had significant advantages in speed and ease-of-use over conventional sequencing, multiple reactions will be required to deliver comparable species-level identification, thus negating many of the benefits of using the technique. We found that 16S rDNA PCR and sequencing should be rationally targeted on the basis of good clinical information in the routine diagnostic setting, and not used as a general screening test for the exclusion of bacterial infection in joint specimens.