Identification of mixed medicinal powder of Epimedii Folium with different origins based on pyrosequencing technology / 药学学报

There are many multi-original medicinal materials in Chinese Pharmacopoeia, and the mixed use of medicinal materials from different sources is common, which has certain influence on the stability of clinical medication. In this study, pyrosequencing technology was used to screen species-specific single nucleotide polymorphisms (SNP) from commonly used DNA barcode sequences, and a rapid and accurate molecular identification method for original species in mixed medicinal powder of Epimedii Folium was established. Multiple sequence alignment analysis showed that the 176th (C/T) mutation and the 196th (A/G) mutation of ITS, the 123rd (C/G) mutation of matK and the 892nd (A/C) mutation of rbcL could be used as the unique SNPs of E. sagittatum, E. koreanum, E. brevicornu and E. pubescens, respectively. In this study, the applicability of pyrosequencing and Sanger sequencing methods in the sequencing of mixture samples was investigated from the perspective of sensitivity and stability. Pyrosequencing method has higher detection sensitivity than Sanger sequencing method for low content samples in the mixed samples. Stability analysis showed that pyrosequencing technology could still obtain effective sequencing results for the amplified products of template DNA after 45 min of 95 ℃ high temperature water bath, while the critical point of Sanger sequencing method was 30 min. In this study, a new identification technology of Epimedii Folium mixed powder primordial species based on pyrosequencing and specific SNP was developed, which can quickly and accurately identify the mixed use of Epimedii Folium with high sensitivity and stability, and can also support the identification of different primordial species and mixed powder primordial herbs, which is conducive to ensuring the consistency and stability of clinical medication.

Methylation status of PRDM12, FOXE1, B3GAT2, VIM and SFRP2 genes in colorectal cancer / 第二军医大学学报

Objective To detect the methylation levels of PR domain-containing protein 12 (PRDM12), forkhead box E1 (FOXE1), beta-1,3-glucuronyltransferase 2 (B3GAT2), vimentin (VIM) and secreted frizzled-related protein 2 (SFRP2) genes in colorectal cancer tissues, so as to evaluate their potentials as early screening markers for colorectal cancer. Methods The paraffin specimen samples were collected from 31 colorectal cancer patients receiving surgical resection in Changhai Hospital, Naval Medical University (Second Military Medical University). The methylation levels of PRDM12, FOXE1, B3GAT2, VIM, SFRP2-1 and SFRP2-2 gene promoters in cancer tissues and corresponding paracancerous normal tissues were detected using pyrosequencing method. Results The promoter methylation indexes of PRDM12, FOXE1, B3GAT2, VIM, SFRP2-1 and SFRP2-2 genes were significantly higher in the cancer tissues of the 31 patients than those in the paracancerous tissues (all P＜0.01). The positive methylation rates of PRDM12, FOXE1, B3GAT2, VIM, SFRP2-1 and SFRP2-2 gene promoters were 87.1% (27/31), 90.3 (28/31), 80.6% (25/31), 77.4% (24/31), 74.2% (23/31) and 64.5% (20/31), respectively. In the 18 cases of early stage (TNM Ⅰ- Ⅱ) colorectal cancer, the positive methylation rates of PRDM12, FOXE1, B3GAT2, VIM, SFRP2-1 and SFRP2-2 gene promoters were 88.9% (16/18), 94.4% (17/18), 83.3% (15/18), 77.8% (14/18), 83.3% (15/18) and 61.1% (11/18), respectively. Conclusion PRDM12 and FOXE1 genes show abnormal methylation in colorectal cancer tissues, suggesting that they may serve as potential molecular markers for the early diagnosis of colorectal cancer.

Quantitative analysis of DNA methylation by pyrosequencing and MassARRAY technique for age estimation: a comparative study / 南方医科大学学报

OBJECTIVE@#To study the difference in age estimation based on quantitative analysis of DNA methylation by MassARRAY and pyrosequencing techniques.@*METHODS@#The methylation levels of 9 CpG sites from two independent whole blood sample sets (containing 65 and 62 samples) were detected using MassARRAY and pyrosequencing techniques. Z-score transformation was used to remove the batch effects of different techniques, and a linear regression model was used for age prediction.@*RESULTS@#For age prediction using the MassARRAY system, the 65 samples showed a mean absolute difference (MAD) of 2.49 years before Z-score transformation of the data and 2.44 years after the transformation, similar to the results in the 62 samples (MAD of 3.36 years before and 3.42 years after Z-score transformation). For data typed from pyrosequencing, the 65 samples showed a MAD of 4.20 years before and 2.76 years after data Z-score transformation, also similar to the results in the 62 samples (MAD of 3.92 years before and 3.63 years after data transformation).@*CONCLUSIONS@#Z-score transformation can effectively reduce the system batch effect between MassARRAY and pyrosequencing. Data from the MassARRAY system allows direct age estimation without further data processing, while the pyrosequencing data may increase the error in age estimation, which can be corrected by Z-score transformation of the data.

Diversidad bacteriana en el biocátodo reductor de Cr(VI) de una Celda de Combustible Microbiana con puente salino / Bacterial diversity in the Cr(VI) reducing biocathode of a Microbial Fuel Cell with salt bridge

Although Cr(VI)-reducing and/or tolerant microorganisms have been investigated, there is no detailed information on the composition of the microbial community of the biocathode microbial fuel cell for Cr(VI) reduction. In this investigation, the bacterial diversity of a biocathode was analyzed using 454 pyrosequencing of the 16S rRNA gene. It was found that most bacteria belonged to phylum Proteobacteria (78.8%), Firmicutes (7.9%), Actinobacteria (6.6%) and Bacteroidetes (5.5%), commonly present in environments contaminated with Cr(VI). The dominance of the genus Pseudomonas (34.87%), followed by the genera Stenotrophomonas (5.8%), Shinella (4%), Papillibacter (3.96%), Brevundimonas (3.91%), Pseu-dochrobactrum (3.54%), Ochrobactrum (3.49%), Hydrogenophaga (2.88%), Rhodococcus (2.88%), Fluviicola (2.35%), and Alcaligenes (2.3%), was found. It is emphasized that some genera have not previously been associated with Cr(VI) reduction. This biocathode from waters contaminated with tannery effluents was able to remove Cr(VI) (97.83%) in the cathodic chamber. Additionally, through use of anaerobic sludge in the anodic chamber, the removal of 76.6% of organic matter (glucose) from synthetic waste water was achieved. In this study, an efficient biocathode for the reduction of Cr(VI) with future use in bioremediation, was characterized.

Aunque se ha investigado sobre los microorganismos reductores y/o tolerantes de Cr(VI), no hay información detallada sobre la composición de la comunidad microbiana del cátodo de una Celda de Combustible Microbiana para la reducción de Cr(VI). En esta investigación se analizó la diversidad bacteriana de un biocátodo usando pirosecuenciación 454 del gen 16S rRNA. Se encontró que la mayoría de las bacterias pertenecieron a los filos Proteobac-teria (78,8%), Firmicutes (7,9%), Actinobacteria (6,6%) y Bacteroidetes (5,5%), comúnmente presentes en ambientes contaminados con Cr(VI). Se encontró como género dominante a Pseudomonas (34,87%), seguido por los géneros Stenotrophomonas (5,8%), Shinella (4%), Papil-libacter (3,96%), Brevundimonas (3,91%), Pseudochrobactrum (3,54%), Ochrobactrum (3,49%), Hydrogenophaga (2,88%), Rhodococcus (2,88%), Fluviicola (2,35%) y Alcaligenes (2,3%). Se destaca que algunos géneros no han sido previamente asociados con la reducción de Cr(VI). Este biocátodo procedente de aguas contaminadas con efluentes de curtiembres fue capaz de remover Cr(VI) (97,83%) en la cámara catódica. Adicionalmente, a través del uso de lodo anaeróbico en la cámara anódica, se logró la remoción del 76,6% de materia orgánica (glucosa) a partir de agua residual sintética. En este estudio se caracterizó un eficiente biocátodo para la reducción de Cr(VI) con futuro uso en biorremediación.

Insights into multiple use water enterobacteria from the Brazilian semi-arid: the first approach by pyrosequencing

Thousands of people living in semi-arid regions face problems of drought and loss of water quality. In addition, high incidence of acute diarrheal diseases related to water consumption has been responsible for a high number of deaths and high economic costs for human health. Many of the diseases can be caused by the presence of enterobacteria in reservoirs that serve for multiple purposes. This study aimed to confirm the presence of potentially harmful bacteria, which was highlighted in other articles, and to reveal non-identified genera by culture-dependent methods and pyrosequencing. Twenty-three genera of the Enterobacteriaceae family were detected, with emphasis on Escherichia genus and confirmation of the presence of species such as Salmonella enterica and Enterobacter cloacae. The abundance of heterotrophic prokaryotes and the physical and chemical data show an expected average for this type of environment due to the numbers historically presented in previous articles. The unprecedented detection of the presence of some potentially pathogenic species can alert and raise awareness of the populations that use stored water in the semi-arid regions. Consequently, as a result of the peculiar characteristics of reservoirs under this climate influence, there is a cosmopolitanism of enterobacteria that may be related to the alarming numbers of infections from Waterborne Diseases.

Establishment of pyrosequencing method for detection of Apolipoprotein E gene polymorphisms / 医学研究生学报

Objective Apolipoprotein E (APOE) is mainly involved in lipid metabolism and cholesterol transport, which is important for health. To establish a pyrosequencing based method for detection of 112T>C and 158C>T single nucleotide polymorphisms (SNPs) in Apolipoprotein E gene. Methods Three amplification systems including Taq enzyme buffer from TaKaRa company (T buffer), La Taq enzyme buffer specified for G-C rich regions (L buffer) and Trans Taq enzyme buffer from TransGen company (TT buffer) were chosen to optimize PCR system and temperature by annealing at 60 °C and gradient annealing from 62 to 68 °C respectively. The specificity of this method was evaluated by comparing its results with those of Sanger sequencing. The sensitivity of this method was evaluated by gradient diluting human genomic DNA as detection template. Results According to the concentration and specificity of the products, the optimum condition was L buffer with 60℃ annealing programs. Pyrosequencing results of 20 samples were completely consistent with those of Sanger sequencing. The sensitivity of this method could be as low as 0.16 ng genomic DNA. Conclusion A method based on pyrosequencing detecting 112 and 158 polymorphisms in APOE gene was established, which can be applied in clinical personalized medicine.

Methylation mechanism of candidate biomarker transcription factor 21 in bladder urothelial carcinoma / 解剖学报

Objective To explore the significance of methylation of the candidate biomarker transcription factor 21(TCF21) in bladder urothelial carcinoma. Methods From October 2016 to October 2017, 142 patients with suspected bladder cancer were selected. Among them, 80 were diagnosed as bladder cancer by pathological examination as the study group. A total of 62 non-bladder cancers were diagnosed by pathological examination as the control group. In addition, 40 healthy urine specimens during the same period were selected as the healthy group. Detected and compared the methylation of TCF21 in bladder cancer tissues, paracancerous tissues, and control tissues of the study group, and detected and compared the TCF21 methylation levels in urine of study group, control group, and healthy group. The relationship between TCF21 methylation level and clinicopathological features was explored, and the diagnostic efficacy of both for bladder cancer was analyzed. Results The methylation level of TCF21 in bladder cancer tissue was significantly higher than that in the adjacent tissue and control group (P 60 years, high TNM stages, and high grade bladder cancer patients (P 0. 05). Conclusion TCF21 gene hasd high methylation level in urine of patients with bladder cancer and bladder cancer, and is associated with pathological features. Urinary bladder cancer tissues and urine have higher diagnostic efficacy for bladder cancer.

Changes in Oral Microbiota in Patients Receiving Radical Concurrent Chemoradiotherapy for The Head and Neck Squamous Cell Carcinoma

Radiotherapy (RT) is a mainstay in the treatment of head and neck squamous cell carcinoma (HNSCC). For locally advanced HCSCC, concurrent chemoradiotherapy (CCRT) benefits HCSCC patients in terms of better survival and loco-regional control. In this study, we evaluated changes in oral microbiota in patients, who received CCRT for head and neck cancer. Oral rinsed samples were weekly collected before and during CCRT and at 4 weeks following treatment from HNSCC patients, who had received 70 Gy of radiation delivered to the primary sites for over 7 weeks and concurrent chemotherapy. Oral microbiota changes in three patients were analyzed by next-generation sequencing using 16S rRNA 454 pyrosequencing. On an average, 15,000 partial 16S rRNA gene sequences were obtained from each sample. All sequences fell into 11 different bacterial phyla. During early CCRT, the microbial diversity gradually decreased. In a patient, who did not receive any antibiotics during the CCRT, Firmicutes and Proteobacteria were the most abundant phylum. During the early CCRT, proteobacteria gradually decreased while Firmicutes increased. During the late CCRT, firmicutes gradually decreased while Bacteroides and Fusobacteria increased. In all the patients, yellow complex showed a gradual decrease, while orange and red complex showed a gradual increase during the CCRT. At 4 weeks after CCRT, the recovery of oral microbiota diversity was limited. During CCRT, there was a gradual increase in major periodontopathogens in association with the deterioration of the oral hygiene. Henceforth, it is proposed that understanding oral microbiota shift should provide better information for the development of effective oral care programs for patients receiving CCRT for HNSCC.

Rapid detection and identification of fungi in urine samples by pyrosequencing / 中华传染病杂志

Objective To establish a rapid method for the clinical detection and identification of fungi in clinical urine samples.Methods DNA was extracted from clinically collected urine sample,and the fungal ribosomal internal transcribed spacer was amplified by polymerase chain reaction (PCR) and followed by pyrosequencing.The fungal species were identified by sequence alignment.Results The identification results were compared between PCR-pyrosequencing and conventional culture method.Among the 1320 urine samples,180 were detected positive by conventional method with the positive rate of 13.6％,while 192 were positive by the pyrosequencing based method with the positive rate of 14.5％.The overall coincidence rate of the two methods was 99.09 ％,with the positive coincidence rate of 100 ％ and the negative coincidence rate of 98.95 ％.The Kappa value was 0.963,suggesting a good consistency.The results of 13 standard strains were consistent with the actual results.Conclusions A rapid culturefree method for the detection of fungi in urine sample has been successfully established.This method is based on PCR-pyrosequencing technology with highly accuracy,sensitivity and reproducibility.It is highly automated,cost effective and with high throughput (96 samples per run).The fungal pathogen in urine is identified by single step test within 3 hours without conventional culture.Thus,it is applicable in the clinical laboratory.

Quantitative analysis of COX-2 promoter methylation in gastric carcinoma

PURPOSE: To determine the occurrence of COX-2 methylation in gastric carcinoma (GC), the status and level of CpG methylation in the promoter region of cyclooxygenase-2 (COX-2) were analyzed in early and advanced GCs, as well as in normal gastric tissues. METHODS: The extent of promoter methylation of the COX-2 gene was assessed quantitatively using pyrosequencing in 60 early and 60 advanced GCs samples harvested upon gastrectomy, and 40 normal gastric mucosa samples from patients with benign gastric diseases as controls. RESULTS: The methylation frequency for the COX-2 gene was significantly higher in early than in advanced GCs (40.0% vs. 20.0%, P < 0.05). A significant difference was found in COX-2 methylation between GCs and normal gastric tissues (30.0% vs. 10.0%, by PS; P < 0.05). COX-2 gene methylation was significantly associated with the depth of invasion (P = 0.003), lymph node metastasis (P = 0.009), distant metastasis (P = 0.036), and TNM staging (P = 0.007). The overall survival of patients with COX-2 methylation was significantly lower than that of patients without COX-2 methylation (P = 0.005). CONCLUSION: These results demonstrated that COX-2 promoter methylation was significantly higher in tumor tissues, and was an early event for GC, thus, COX-2 gene methylation may be important in the initial development of gastric carcinogenesis. Thus, GCs with methylation in COX-2 may not be good candidates for treatment with COX-2 inhibitors. Furthermore, COX-2 methylation could be a significant prognostic factor predicting a favorable effect on GC patient outcome when downregulated.

Application of the pyrosequencing technique for detection of VKORC1 and CYP2C9 genotypes / 国际检验医学杂志

Objective To establish genotyping methods for vitamin K epoxide reductase complex subunit 1 (VKORC1)and cytochrome P450 2C9(CYP2C9)based on pyrosequencing technique to detection of warfarin metabolizing enzyme related gene polymorphisms.Methods A total of 50 peripheral blood samples from healthy adults were collected and the whole blood genomic DNA was extracted.A set of biotin-labeled amplifi-cation primers and sequencing primers were designed respectively for three SNP sites:VKORC1 -1639 G>A,CYP2C9 430C> T and CYP2C9 1075A>C.After PCR amplification of the samples,pyrophosphoric acid se-quencing was conducted.And then the signal peaks form were combined to analyze and determine each sample genotype.Genotyping results were verified by Sanger sequencing,and the consistency of the two sequencing methods was compared.Results Genotypes of the three SNPs can be clearly determined according to the ba-ses and height of the signal peaks.Among the 50 samples,there were 41 AA and nine AG for VKORC1 -1639G>A,accounting for 82% and 12% respectively,and there were 45 *1/*1,five *1/*3 for CYP2C9, accounting for 90% and 10% respectively,no CYP2C9*2 allele detected.Genotype results detected by pyrose-quencing and Sanger sequencing were consistent with each other.Conclusion In SNP genotyping,Pyrose-quencing has the advantages of convenience,time-saving,cheap with accurate and reliable results,which can quickly determine the genotypes of CYP2C9 and VKORC1.

Guild Patterns of Basidiomycetes Community Associated With Quercus mongolica in Mt. Jeombong, Republic of Korea

Depending on the mode of nutrition exploitation, major fungal guilds are distinguished as ectomycorrhizal and saprotrophic fungi. It is generally known that diverse environmental factors influence fungal communities; however, it is unclear how fungal communities respond differently to environment factors depend on fungal guilds. In this study, we investigated basidiomycetes communities associated with Quercus mongolica using 454 pyrosequencing. We attempted to detect guild pattern (ectomycorrhizal or saprotrophic fungal communities) by comparing the influence of geography and source (root and surrounding soil). A total of 515 mOTUs were detected from root (321) and soil (394) of Q. mongolica at three sites of Mt. Jeombong in Inje County. We found that patterns of diversity and community structure were different depending on the guilds. In terms of alpha diversity, only ectomycorrhizal fungi showed significant differences between sources. In terms of community structure, however, geography significantly influenced the ectomycorrhizal community, while source appeared to have a greater influence on the saprotrophic community. Therefore, a guild-based view will help to elucidates novel features of the relationship between environmental factors and fungal communities.

Development and characterization of microsatellite markers in Gaultheria pumila Lf. (Ericaceae)

BACKGROUND: Polymorphic microsatellite markers were developed for Gaultheria pumila (Ericaceae) to evaluate genetic diversity and population structure within its native range in Chile. This is a very important Ericaceae endemic to Chile with a large commercial potential. Its resistance to different abiotic conditions makes it a valuable target for genetic improvement. RESULTS: Ten polymorphic simple sequence repeat (SSR) loci were isolated from Gaultheria pumila using new-generation 454 FLX Titanium pyrosequencing technology. The mean number of alleles per locus ranged from 2 to 4. Observed and expected heterozygosity ranged from 0.00 to 1.0 and 0.00 to 0.64, respectively. CONCLUSIONS: From 10 SSR markers developed for G. pumila, 9 markers are promising candidates for analyzing genetic variation within or between natural populations of G. pumila and other species from the same genus.

TaqMan Real-time PCR,pyrosequencing and GeneChip microarray using the SNP protocol for rapid determination of ALDH2*2 in a Chinese population / 西安交通大学学报(医学版)

ABSTRACT:Objective To establish a rapid molecular method for the detection of aldehyde dehydrogenase-2 (ALDH2)and investigate the gene polymorphisms of ALDH2?2 and determine whether the polymorphic ALDH2 gene is associated with drinking behavior in a Chinese population.Methods The gene polymorphisms of ALDH2?2 were detected using pyrosequencing,TaqMan Real-time PCR and GeneChip microarray technologies;genotyping of 302 volunteers was performed to assess their genetic associations with alcohol use behavior.Results We developed pyrosequencing,TaqMan Real-time PCR and GeneChip microarray methods to identify ALDH2? 2 polymorphisms.The allele frequency of ALDH2?2 was 20.36% in the Chinese population:16.33% in the alcoholic group and 27.83% in non-drinkers (P=0.001).In contrast,the genotype frequency of heterozygous ALDH2?1/?2 plus homozygous ALDH2?2/?2 was 45.28% in non-drinkers and 32.65% in the alcoholics group (P=0.030). Allele frequency of ALDH2 genotypes differed significantly between our Chinese sample and other ethnic groups in Asia,and it was significantly higher than that in European and American countries.Conclusion The developed pyrosequencing,TaqMan Real-time PCR and GeneChip microarray methods are rapid,accurate,high-throughput, convenient,and reliable for detecting ALDH2 polymorphisms.ALDH2?2 gene can protect against the development of alcoholism.The allele frequency of ALDH2 in this Chinese population differs from that in other ethnic groups.

Genotype method for SNPs related to individualized aspirin treatment based on pyrosequencing technology / 中国药科大学学报

There are significant individual differences in the antiplatelet effects of aspirin.Three single nucleotide polymorphisms (SNPs),rs5918,rs12041331 and rs730012,are reported to significantly correlate with the efficacy and side effects of aspirin.In the present study,the genotyping method of the three SNPs was established based on the combination of polymerase chain reaction and pyrosequencing technology.Amplification and sequencing primers were designed independently;the amplification conditions were optimized to amplify the three SNPs in the same condition.The sensitivity of the method was detected using original genome DNA at different concentrations.In order to testify the accuracy of the method,the proposed method and Sanger sequencing technology were both used to genotype the three SNPs in 20 blood samples.The results demonstrated that the genotyping method of aspirin-related SNPs was successfully established,with the detection limit being as low as 0.4 ng genome DNA.The genotype results of 20 samples by the proposed method were exactly the same as that of Sanger sequencing.It is evident that the proposed method is sensitive and accurate.

Establishment of a PCR-pyrosequencing method for the rapid detection of three marine vibrios and the investigation on 16S rRNA genotyping of Vibrio vulnificus / 临床检验杂志

Objective To establish a rapid diagnostic method for the detection of marine vibrios,and then construct a new technology platform for the clinical diagnosis of marine vibrio infection.Methods A pair of PCR primers and a sequencing primer based on the whA gene of V.vulnificus and the toxR genes of V.parahemolyticus and V.alginolyticus were designed respectively,and then the specific DNA fragments were amplified.Next,the single-stranded DNA templates were prepared for pyrosequencing.The obtained base sequence was validated by NCBI alignment.In addition,the 16S rRNA genotyping of V.vulnificus was also performed.Results The PCR primers and sequencing primer of V.vulnificus showed good specificity,and a 167 bp DNA fragment was amplified from 4 strains of V.vulnificus.The pyrosequencing results completely matched with the whA gene sequence of V.vulnificus.Meanwhile,the control strains were negative.A 105 bp DNA fragment and a 134 bp DNA fragment were amplified from 11 strains of V.parahemolyticus and V.alginolyticus,respectively,and the pyrosequencing results were consistent with the expected sequence.In addition,one of 4 strains of V.vulnificus was identified as 16S rRNA-A type,and the other 3 as 16S rRNA-B type.Conclusion The PCR-pyrosequencing method established in this study is a new method for the real-time detection of short nucleotide sequences.It has some advantages such as high throughput,high precision and simple operation,and may be applied to the fast and accurate identification of marine and terrestrial pathogenic bacteria.

Bacterial diversity and its relationship to growth performance of broilers

The microbial community is known to have a key role during the rearing period of broilers. In this study, gut microbial composition and diversity were examined to evaluate the relationships between these factors and broiler growth performance. By applying 454-pyrosequencing of the V1–V3 regions of bacterial 16S rRNA genes, six fecal samples from four- and 28-day-old chickens from three broiler farms and 24 intestinal samples of broilers with heavy and light body weights were analyzed. Microbial composition assessment revealed Firmicutes to be the most prevalent phylum at farm A, while Proteobacteria were predominant at farms B and C. Fecal microbial richness and diversity indices gradually increased from four to 28 days at all three farms. Microbial diversity assessment revealed that small intestine microbial diversity was lower in heavy birds than in light birds. In light birds, the Firmicutes proportion was lower than that in heavy birds. In conclusion, each broiler farm revealed a specific microbial profile which varied with the age of the birds. The microbial communities appeared to affect growth performance; therefore, gut microbial profiles can be utilized to monitor growth performance at broiler farms.

KRAS Mutation Test in Korean Patients with Colorectal Carcinomas: A Methodological Comparison between Sanger Sequencing and a Real-Time PCR-Based Assay

BACKGROUND: Mutations in the KRAS gene have been identified in approximately 50% of colorectal cancers (CRCs). KRAS mutations are well established biomarkers in anti–epidermal growth factor receptor therapy. Therefore, assessment of KRAS mutations is needed in CRC patients to ensure appropriate treatment. METHODS: We compared the analytical performance of the cobas test to Sanger sequencing in 264 CRC cases. In addition, discordant specimens were evaluated by 454 pyrosequencing. RESULTS: KRAS mutations for codons 12/13 were detected in 43.2% of cases (114/264) by Sanger sequencing. Of 257 evaluable specimens for comparison, KRAS mutations were detected in 112 cases (43.6%) by Sanger sequencing and 118 cases (45.9%) by the cobas test. Concordance between the cobas test and Sanger sequencing for each lot was 93.8% positive percent agreement (PPA) and 91.0% negative percent agreement (NPA) for codons 12/13. Results from the cobas test and Sanger sequencing were discordant for 20 cases (7.8%). Twenty discrepant cases were subsequently subjected to 454 pyrosequencing. After comprehensive analysis of the results from combined Sanger sequencing–454 pyrosequencing and the cobas test, PPA was 97.5% and NPA was 100%. CONCLUSIONS: The cobas test is an accurate and sensitive test for detecting KRAS-activating mutations and has analytical power equivalent to Sanger sequencing. Prescreening using the cobas test with subsequent application of Sanger sequencing is the best strategy for routine detection of KRAS mutations in CRC.

Influence of oil pollution on soil microbial community diversity / 生物工程学报

Bacteria, actinomycetes and fungi are the three major groups of soil microbes. Soil microbes play a critical role in ecological and biodegradation processes in petroleum-contaminated soils. Based on the actual situation, this study took the oil polluted soil around the abandoned oil well in Shehong County, Suining City, Sichuan Province as the test soil. First, we determined the physiochemical properties of the tested soil; then we analyzed the changes of physiochemical properties and the three major microbes in petroleum contaminated soils. The number of the three major microbes in contaminated soils was relatively fewer than uncontaminated samples, and the water content of the soil was in positive correlation with the number of microbes. Also we assessed the soil bacteria community diversity and changes therein in petroleum-contaminated soils using 454 pyrosequencing of 16S rRNA genes. No less than 23 982 valid reads and 6 123 operational taxonomic units (OTUs) were obtained from all 4 studied samples. OTU richness was relatively higher in contaminated soils than uncontaminated samples. Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Planctomycetes and Proteobacteria were the dominant phyla among all the soil samples. However, the prokaryotes community abundance of phyla was significantly different in the four samples. The most abundant OTUs associated with petroleum-contaminated soil sample were the sequences related to Acidobacteria, Actinobacteria and Proteobacteria, whereas the most abundance sequences with uncontaminated sample were those related to Actinobacteria, Bacteroidetes and Proteobacteria.

Microbiota bacteriana asociada al papel moneda de circulación en Colombia / Bacterial microbiota on circulating Colombian banknotes

Diariamente los seres humanos están en interacción con objetos de uso continuo, como el papel moneda, sin el conocimiento de que estos almacenan microorganismos y de que nos exponemos al contacto con potenciales patógenos. La composición de la comunidad bacteriana en un billete colombiano fue determinada mediante el secuenciamiento profundo de librerías de amplicones 16S. Se encontraron 233 géneros bacterianos; 12 de estos géneros corresponden a especies con potencial patogénico. El género más abundante fue Propionibacterium, seguido de Streptococcus, Staphylococcus Pseudomonas . Este es el primer reporte de la diversidad bacteriana que puede ser alojada en este objeto de alta circulación en Colombia. Pocos estudios en el mundo han mostrado este nivel de detalle de la microbiota en billetes de circulación y ofrece un panorama mucho más amplio de la exposición diaria a microorganismos al utilizar papel moneda en las condiciones en las que se utiliza en Colombia.

Commonly used objects such as currency paper can be colonised by bacteria and can serve as carriers of microbes. This colonisation might expose us to unnoticed pathogenic bacteria. In this study, the researchers obtained a detailed panorama of the microbes that can be carried on currency notes in Colombia by using 454 next-generation deep sequencing of 16S amplicón libraries. A total of 233 bacterial genera were detected and classified, 12 of which are potential human pathogens. The most abundant genera were Propionibacterium, Streptococcus, Staphylococcus and Pseudomonas. To date, this is the first in-depth analysis of the microbiota carried by circulating banknotes in our continent and it offers insights into daily exposure to microbes when using banknotes in Colombia.