Establishment of Molecular Biological Method for Identification of Bacteria by 16S rDNA and gyrB Gene

[Objectives]The paper was to establish a molecular biological method for identification of bacterial strains.[Methods]The thalli of standard bacterial strains existing in the laboratory were collected and genomic DNA was extracted for amplification of 16S rDNA and gyrB gene.The 16S rDNA and gyrB gene sequences were obtained after sequencing.Sequences were aligned and analyzed via EzBioCloud and NCBI database,and phylogenetic trees were constructed to determine the species relationship of strains.Meantime,they were compared with known strains.[Results]This method could identify 5 standard strains accurately to the species level.The 16S rDNA and gyrB gene sequences were aligned and analyzed in EzBioCloud database and NCBI database.The strain with the max score was consistent with the known strain.And the query cover and ident were both above 99%.[Conclusions]The established molecular biological method for identification of bacterial strains by 16S rDNA and gyrB gene has good accuracy,which effectively solves the problem that the laboratory identification of bacteria relies on traditional methods and the accuracy can not be guaranteed,and further improves the identification ability of laboratory bacterial strains.

奶牛瘤胃需氧及兼性厌氧菌的PCR-16S rDNA鉴定及日粮的影响

设计9个精料梯度的日粮饲喂干乳期成年荷斯坦奶牛,并对其瘤胃液中的部分细菌进行了分离,PCR-16SrDNA鉴定和动态检测。结果显示,以瘤胃液为原料,采用人工培养基共分离到56株细菌,并对7株具代表性的细菌进行了PCR扩增,经序列同源性比较,最终把细菌鉴定为牛链球菌(A0625、A1212、0131)、蜡样芽孢杆菌(0113)、地衣芽孢杆菌(0091)、短小芽孢杆菌(0083)和嗜热链球菌(0123)。同时还发现随精料浓度的增加,牛链球菌、芽孢杆菌及嗜热链球菌的数量均呈不同程度的上升趋势。结果表明不同精料浓度的日粮对奶牛瘤胃需氧及兼性厌氧菌有着不同程度的影响,其中对牛链球菌影响最大,从系统发育分析可知所分离细菌虽未形成新的细菌进化枝,但牛链球菌和嗜热链球菌的核苷酸序列仍发生了多处碱基突变。

小叶锦鸡儿根瘤菌的分离及其16S rDNA PCR-RFLP分析

对辽宁地区与小叶锦鸡儿共生的根瘤菌资源进行了初步调查。采自5个不同地区样品的根瘤,通过分离、纯化、回接验证等试验共获得65株供试根瘤菌菌株。进一步选用4种限制性内切酶对供试根瘤菌进行了16S rDNA PCR-RFLP研究,结果表明其系统发育地位位于中慢生根瘤菌属(Mesorhizobiumspp.),并在96%相似性水平上分为5个不同的类群,分别由相应的rDNA图谱组合代表。丰富度及频度分析表明,组合15是辽宁省的优势群,组合18丰富度居第二位,但频度最高,也是辽宁省的主要类群。

草本纤维提取用菌株的PCR-16S rDNA及ITS-RFLP分析

通过分析从沤麻水中分离到的8株草本纤维提取用菌株的16S rDNA以及ITS序列,鉴定并构建其聚类图,明确草本纤维提取用菌株的遗传关系。通过BLAST比对16S rDNA并用限制性内切酶酶切16～23S的ITS保守序列,对参试菌株的16S rDNA及ITS酶切片段进行聚类分析。结果表明,8株菌株分别属于地衣芽孢杆菌、梭状芽孢杆菌、蜡状芽孢杆菌、短小芽孢杆菌、枯草芽孢杆菌以及多粘类芽孢杆菌;基于16S rDNA序列与ITS-RFLP所获得的聚类结果基本一致,菌株1251与菌株1172-1聚为一类,其余聚为一类,但种内聚类存在一定的差异,可能与不同类型的序列进化程度有关,8株菌株的16SrDNA序列的相似性为78.66%;AluⅠ、HinfⅠ、Sau3AⅠ、TaqⅠ4种限制性内切酶对参试的8株草本纤维提取用产芽孢菌株的ITS进行酶切,各获得0～4条100～440bp的酶切产物。

Association between childhood obesity and gut microbiota:16S rRNA gene sequencing-based cohort study

BACKGROUND This study aimed to identify characteristic gut genera in obese and normal-weight children(8-12 years old)using 16S rDNA sequencing.The research aimed to provide insights for mechanistic studies and prevention strategies for childhood obesity.Thirty normal-weight and thirty age-and sex-matched obese children were included.Questionnaires and body measurements were collected,and fecal samples underwent 16S rDNA sequencing.Significant differences in body mass index(BMI)and body-fat percentage were observed between the groups.Analysis of gut microbiota diversity revealed lowerα-diversity in obese children.Differences in gut microbiota composition were found between the two groups.Prevotella and Firmicutes were more abundant in the obese group,while Bacteroides and Sanguibacteroides were more prevalent in the control group.AIM To identify the characteristic gut genera in obese and normal-weight children(8-12-year-old)using 16S rDNA sequencing,and provide a basis for subsequent mechanistic studies and prevention strategies for childhood obesity.METHODS Thirty each normal-weight,1:1 matched for age and sex,and obese children,with an obese status from 2020 to 2022,were included in the control and obese groups,respectively.Basic information was collected through questionnaires and body measurements were obtained from both obese and normal-weight children.Fecal samples were collected from both groups and subjected to 16S rDNA sequencing using an Illumina MiSeq sequencing platform for gut microbiota diversity analysis.RESULTS Significant differences in BMI and body-fat percentage were observed between the two groups.The Ace and Chao1 indices were significantly lower in the obese group than those in the control group,whereas differences were not significant in the Shannon and Simpson indices.Kruskal-Wallis tests indicated significant differences in unweighted and weighted UniFrac distances between the gut microbiota of normal-weight and obese children(P<0.01),suggesting substantial disparities in both the species and quantity of gut microbiota between the two groups.Prevotella,Firmicutes,Bacteroides,and Sanguibacteroides were more abundant in the obese and control groups,respectively.Heatmap results demonstrated significant differences in the gut microbiota composition between obese and normal-weight children.CONCLUSION Obese children exhibited lowerα-diversity in their gut microbiota than did the normal-weight children.Significant differences were observed in the composition of gut microbiota between obese and normal-weight children.

Monitoring and identification of spoilage-related microorganisms in braised chicken with modified atmosphere packaging during refrigerated storage

This study mainly monitored the dominant bacterial populations and identified the spoilage-related microorganisms of braised chicken meat stored under different CO_(2)-modified atmosphere packaging(MAP)during refrigerated storage using a culture-dependent method and 16S rDNA identification.The quality changes and shelf life of the meat were also measured.The growth rate of total viable count(TVC)in braised chicken was slower with an increase of CO_(2) content in MAP,which also occurred in the remaining bacterial species monitored(lactic acid bacteria,Pseudomonas spp.,Brochothrix thermosphacta).The MAP exerted beneficial effects on the quality of braised chicken,as demonstrated by retarding the production of total volatile basic nitrogen(TVB-N)and delaying lipid oxidation(TBARS test).A total of 14 isolates were identified from braised chickens with different packaging at the end of storage,these included P.fragi(6 isolates),P.psychrophila(2 isolates),Enterococcus faecalis(3 isolates),B.thermosphacta(2 isolates),Staphylococcus equorum(1 isolate).

牦牛肠道与粪便乳酸菌的分离鉴定及PCR-16 S rDNA鉴定

以取自四川省不同地区的牦牛粪便、肠道内容物为材料,用MRS琼脂双层培养基进行厌氧培养,分离到50株乳酸菌,经生化鉴定为嗜热链球菌(2株)、乳酸乳球菌(1株)、保加利亚乳杆菌(5株)、嗜粪乳杆菌(10株)、嗜酸乳杆菌(8株)、乳酸乳杆菌(9株)、肠乳杆菌(10株)、弯曲乳杆菌(5株)。采用乳酸菌16 S rDNA通用引物,对分离的8种菌的16 S rDNA一段可变区序列进行扩增,均得到大小约470 bp的产物;扩增产物经纯化、测序后与GenBank中标准菌株的核甘酸序列比较,同源性均大于97．5％,同源性分析与生化试验的结果是一致的。证实,牦牛肠道和粪便的乳酸菌较为丰富,且乳杆菌的数量较多,这可能与牦牛复杂的生长环境有关。

利用16S rDNA序列对两种芽孢杆菌的鉴定

根据不同种属细菌的16 S rDNA序列两端的保守性区域设计通用引物,提取菌株的基因组DNA,对菌株的16 S rDNA进行了PCR扩增,对扩增到的目标片段进行了测序,将测序结果与NCBI上已知菌种的16 S rDNA序列进行了BLAST对比,并构建了系统进化树。结合传统的形态观察及生理生化特性鉴定,16 S rDNA序列分析结果证实芽孢杆菌Pab02为枯草芽孢杆菌,PAS38为蜡样芽孢杆菌。

致鹅卵黄性腹膜炎大肠杆菌的分离与16SrDNA的鉴定

从鹅卵黄性腹膜炎临床病例中采集样品,分离病原进行一系列生化鉴定,共分离得到7株致鹅卵黄性腹膜炎大肠杆菌,编号为E0238、E0239、E0240、E0453、E0454、E0241、E0245。O血清型鉴定分属于O2、O21、O37、O1、O24、O24、O148。利用16SrDNA扩增试剂盒对随机选取的3株细菌进行PCR扩增,并将PCR产物序列与GenBank中的E.coli16SrDNA进行序列比对,结果显示分离细菌与大肠杆菌的同源性达99%以上。

一株益生芽孢杆菌Pab02的16S rDNA测序鉴定

利用16S rDNA分析对Pab02芽孢杆菌型益生菌进行系统进化鉴定。首先提取菌株Pab02的基因组DNA,根据不同种属细菌的16S rDNA序列两端的保守性设计通用引物,对菌株Pab02的16S rDNA的进行PCR扩增,并对扩增到的目标片段进行测序,将测序结果与NCBI上已知菌种的16S rDNA序列进行BLAST对比,初步构建系统进化树进行分析,再结合传统的形态观察及生理生化特性综合鉴定,最终确定为枯草芽孢杆菌。

Application of 16S rDNA Sequence Analysis Technique in Microbial Detection

With conserved regions and regions of high variations, 16s rDNA is an important molecular basis for the biological species identification and system evolu- tion. The modem molecular biology with 16s rDNA as the primer can accurately re- veal the diversity of microorganisms species and inheritance, thereby 16s rDNA se- quence analysis has become the main basis for classification and identification of microorganisms. Having overcome the limitations of traditional microculture methods, this method is easy to operate, quick and accurate to detect with high sensitivity, making it widely apply to species identification, community comparative analysis, phytecoenogenesis and the assessment of population diversity. It is a objective classification method with high credibility.

降解烟碱的YC-68菌株16 S rDNA序列分析及鉴定

从烟叶调制过程中温度达68℃阶段的叶片上分离得到一株能降解烟碱的菌株,编号为YC-68,该菌经常规的形态、生理生化分析,确定为芽孢杆菌。根据该菌16 S rDNA恒定区的保守性设计了一对通用引物,扩增其16 S rDNA序列,将扩增的产物进行回收后进行测序,把测序后的结果提交到GenBank利用BLAST进行序列同源性分析。经过分析鉴定YC-68菌株为芽孢杆菌属的蜡状芽孢杆菌。

基于16S rDNA序列的4株气单胞菌属细菌的分子鉴定

对分离自养殖水环境的4株气单胞菌属(Aeromonas)细菌的16 S rDNA序列进行PCR扩增并测定其核酸序列,通过BLAST软件在GenBank中查找同源序列,应用MegAlign软件中的Jotun Hein、ClustalV以及ClustalW 3种方法进行序列差异和同源性分析,分别使用Mega 4.0软件中的邻接法(N-J)、最小进化法(ME)、最大简约法(MP)、非加权组平均法(UPGMA)构建系统发育树。由Jotun Hein法可知,Aeromonas sp.T3与序列DQ817542.1、DQ817645.1以及HM127065.1相似度最高,均为99.2%,Aeromonas sp.T4与序列DQ816364.1、HM77846.1以及HM778618.1相似度最高,均为97.9%,Aeromonassp.T5与序列GQ205446.1相似度最高,均为99.4%,Aeromonas sp.T6与序列GQ205446.1相似度最高,为99.6%;其他两种方法的结果相似度略低。4种方法构建的系统发育树基本一致,可初步确立4株菌株的分类地位:Aeromonas sp.T3与序列DQ817542.1亲缘关系最近,Aeromonas sp.T4与序列HM778618.1亲缘关系最近,Aeromonas sp.T5与序列GQ232759.1亲缘关系最近,Aeromonas sp.T6与序列GQ205446.1亲缘关系最近。

拮抗放线菌111A202的种类鉴定及其16S rDNA序列分析

对分离自药用植物假橐吾的一株拮抗放线菌菌株111A202进行了形态特征、培养特征、生理生化特征、细胞壁组分分析及16S rDNA序列分析。结果表明该菌基内菌丝无横隔、不断裂,气生菌丝分枝、孢子丝波曲,孢子椭圆形,表面光滑。细胞壁化学组分Ⅰ型。以16S rDNA序列为基础构建了包括7株相关种属菌在内的系统发育树,111A202与变铅青链霉菌(Streptomyces lividans)、微白黄链霉菌(S.albido-flavus)的16S rDNA序列的相似性达到99.5%和99.6%。根据以上多相分类结果,放线菌111A202的形态培养特征及其细胞组分与链霉菌一致,且与微白黄链霉菌近似性很高,因此,可以将放线菌111A202定名为微白黄链霉菌111A202(S.albidoflavus 111A202)。

Isolation and Identification of High-Quality Lactic Acid Bacteria in Forage Corn

[ Objective] To screen suitable lactic acid bacterium strains from forage corn which can be used as silage additives. [ Method] The lactic acid bacterium strains were isolated by inoculation on MRS solid medium containing calcium carbonate, and they were preliminarily identified through morphological, physiological and biochemical experiments. The acid production efficiency was determined. Twelve strains having strong acid-pro- duction ability were selected, and their salt tolerance and acid tolerance were detected. The sequences of their 16 S rDNA were also analyzed. [ Result] A total of 44 lactic acid bacterium strains were isolated from the forage com. As evidenced by the physiological and biochemical experi- ments, the twelve strains having strong acid-production ability belonged to Leuconostoc, Lactobacillus and Enterococcus, respectively, and they had strong salt tolerance and acid tolerance. According to the sequences of 16 S rDNA, A4, B9, B11, B12 and B14 were Lactobacillus plantarum; A1, A2., A7, A11 and B8 were Leuconostoc mesenteroides dextran subspecies; and AB and A9 were Enterococcus hirae. [ Conclusion ] The lactic acid bacterium strains with strong acid-production ability isolated from forage corn can be developed into silage additives.

Genetic Relationships of Soft Rot Bacteria Isolated from Konjac in China by Amplified Fragment Length Polymorphism (AFLP) and 16S rDNA Gene Sequences

Twenty-three isolates of soft rot bacteria from konjac corms were examined for their diversity using 16S rDNAs and AFLP technology. Both methods clustered two groups, dependent on their biotype characterization of Pectobacterium carotovora subsp. carotovora (P.c.c) and Pectobacterium chrysanthemi (P.ch), respectively. Of all isolates, 17 (73.9%) belonged to P. ch, indicated as the main pathogenic bacteria of konjac producing areas in China. The genetic variation among isolates from the same biotype was also rich, not consistent with the distances of the geographic sources.

Isolation,Screening and Identification of Antagonistic Actinomycetes against Ustilago scitaminea Syd

[Objectives] This study was conducted to obtain actinomycetes strains having antagonistic effect on Ustilago scitaminea Syd.[Methods] At first, actinomycetes strains were isolated from 22 soil samples in Hainan sugarcane regions. Then, antagonistic actinomycetes against U. scitaminea were screened by confrontation culture. Finally, the taxonomic status of antagonistic actinomycetes was determined using 16S rDNA.[Results] From the 22 samples, 984 actinomycetes strains were isolated. From all the isolated strains, 23 antagonistic actinomycetes strains were obtained through primary screening, and one strains with better antagonistic effect was then obtained through secondary screening, and designated FAS. 16S rDNA identification showed that strain FAS shared 99% sequence similarity with Streptomyces cealestis US24. A phylogenetic tree was built with MAGE 7.0 software, and the results showed that strain FAS had the shortest genetic distance with S. caelestis US24. Therefore, the actinomycetes FAS was determined as S. caelestis .[Conclusions] This study provides a new biocontrol method for the biological control of sugarcane smut, thereby ensuring sustainable development of sugarcane industry and sugar industry.

Isolation and Identification of Cellulose- decomposing Bacteria from Deep-litter Systems

In this study, 43 cellulose-decomposing strains were isolated from deep-litter systems. After preliminary screening with Congo red identification medium and filter paper strip medium, five strains with large transparent circles that disintegrated filter paper strips were obtained. After further liquid fermentation, CMC activity, FPA activity and natural eellulase activity of these five strains were determined, and two cellulose-decomposing strains with higher enzyme activity were screened, which were named F7 and F21. Based on molecular biological identification and phylogenetic analysis of 16S rRNA gene sequences, these two cellulose- decomposing strains were identified as Bacillus subtilis and Streptomyces sp. , respectively.

两株产生大环内酯Macrolactin S的海洋细菌的筛选及鉴定

目的:从中国东海分离筛选抗菌活性微生物,确定活性菌株的分类地位。方法:随机挑选30株本室保存的分离自中国东海的细菌,利用抗大肠杆菌模型进行活性筛选。对活性菌株进行形态特征、生化特征、盐需求测试及16S rDNA序列分析,并将所测得的序列在NCBI数据库进行相似性搜索,选取其中具有代表性的序列以邻接法构建系统进化树,将菌株鉴定到属。结果:筛选出活性菌株F81612和F201721,它们的最适生长盐浓度分别为10%和7.5%,菌株形态特征、生化性质与Bacillus sp.相符,菌株F81612和F201721的16S rDNA序列分别与Bacillus subtilis和Bacillus amyloliquefaciens相似程度最大。结论:利用抗大肠杆菌模型筛选到两株产大环内酯Macrolactin S的细菌,均为中等嗜盐的海洋Bacillus sp.。

Integrating a DNA barcoding project with an ecological survey:a case study on temperate intertidal polychaete communities in Qingdao,China

In this study,we integrated a DNA barcoding project with an ecological survey on intertidal polychaete communities and investigated the utility of CO1 gene sequence as a DNA barcode for the classification of the intertidal polychaetes.Using 16S rDNA as a complementary marker and combining morphological and ecological characterization,some of dominant and common polychaete species from Chinese coasts were assessed for their taxonomic status.We obtained 22 haplotype gene sequences of 13 taxa,including 10 CO1 sequences and 12 16S rDNA sequences.Based on intra-and inter-specific distances,we built phylogenetic trees using the neighbor-joining method.Our study suggested that the mitochondrial CO1 gene was a valid DNA barcoding marker for species identification in polychaetes,but other genes,such as 16S rDNA,could be used as a complementary genetic marker.For more accurate species identification and effective testing of species hypothesis,DNA barcoding should be incorporated with morphological,ecological,biogeographical,and phylogenetic information.The application of DNA barcoding and molecular identification in the ecological survey on the intertidal polychaete communities demonstrated the feasibility of integrating DNA taxonomy and ecology.