虫草属真菌的ITS序列分析和系统发育研究

收集不同产地虫草属真菌,进行ITS(Internal Transcribed Spacer)区段克隆测序和序列特征比较分析,并对ITS序列进行核酸序列数据库GenBank同源性检索比对,并与检索获得的7个最相似物种的ITS序列构建系统发育树.结果表明,虫草属真菌的ITS序列长度为570～576bp,GC含量56.29～56.77%,系统发育分析显示,供试菌株中除C-6与九州虫草高度同源外,其他与蛹虫草均具有高度的亲缘关系.     

石斛属植物DNA条形码鉴定及铁皮石斛产地分析

为建立石斛属植物适合的DNA条形码以及利用条形码鉴定铁皮石斛产地，对鼓槌石斛、报春石斛、金钗石斛、铁皮石斛4种石斛以及杭州、霍山、云南和温州4个产地的铁皮石斛的66份样品的ITS2和psbA-trnH序列进行扩增、测序及序列分析。比较石斛属植物种内、种间的变异，基于K2P距离分析Barcoding Gap，构建NJ系统发育树。结果表明，ITS2序列种内遗传变异(0～0.005)小于种间遗传变异(0.070～0.228)，NJ进化树也能准确区分4种石斛；而psbA-trnH序列未形成Barcoding Gap，NJ进化树未能区分报春石斛和金钗石斛，psbA-trnH序列在鉴定石斛属植物时可作为补充序列。ITS2序列和psbA-trnH序列在4种产地铁皮石斛中存在多个变异位点，可鉴别铁皮石斛产地。     

基于ITS序列分析对疑似白羊肚菌株的分子鉴定

通过提取疑似白羊肚菌株基因组DNA,采用通用引物ITS1和ITS4扩增ITS序列,利用GenBank中的BLAST软件搜索同源序列进行比对,并构建系统发育树,对疑似菌株白羊肚进行了初步分子鉴定。结果表明,在GenBank数据库中登录的有26 个菌株与白羊肚ITS 序列相似度为99%,其中Pleurotus eryngii (杏鲍菇)15 个,P. nebrodensis(白灵菇)11 个。根据rDNA ITS 区序列分析准则和白羊肚菌褶呈网格状,菌盖为白色的形态特征,确定疑似白羊肚菌株为与白灵菇(P. nebrodensis)和杏鲍菇(P. eryngii)高度近缘的一个菌株。     

基于GC-MS建立花椒挥发油指纹图谱及在汉源红花椒鉴定中的应用

本研究建立了我国西部地区红花椒和青花椒挥发油气相色谱一质谱(GC-MS)指纹图谱,并将该方法应用到汉源红花椒产地鉴别中。采用水蒸气蒸馏法提取花椒挥发油,通过GC-MS技术建立花椒挥发油指纹图谱,通过挥发油成分差异分析、聚类分析法和主成分分析法对花椒样品进行分类分析。结果表明,青花椒挥发油含量是红花椒挥发油含量的1.45倍。挥发油主要成分为萜烯类、醇类和酯类物质。红花椒指纹图谱有8个共有峰,青花椒指纹图谱有11个共有峰。聚类分析和主成分分析结果相似,所有的青花椒被分为同一组,红花椒被分为2～3组,说明不同产地红花椒挥发油化学成分之间的差异性大于青花椒。汉源红花椒指纹图谱共有峰中有5种特有物质,即崖柏烯,乙酸松油酯,乙酸橙花酯,乙酸香叶酯和1-石竹烯,可作为汉源红花椒产地鉴别的依据。本实验为不同产地花椒产地鉴定提供了实验基础,为汉源花椒建立了特征指纹图谱。     

基于ITS和IGS1序列分析对云南牛肝菌的分类鉴定

以采自云南地区的5个牛肝菌(Boletus sp.)样品为材料,根据样品的形态特征,并结合r DNA ITS和IGS1序列分析对5个样品进行分类鉴定,通过与Gen Bank上已登录的牛肝菌序列比对,采用邻接法(neighbor-joining,N-J)构建系统发育树。结果表明,所有供试材料的ITS和IGS1全长分别在739~787bp和537~583bp范围内,GC含量分别在48.31%~51.01%和48.42%~52.89%之间,遗传距离在0.017~0.248和0.002~0.225之间,上述数据表明云南5个牛肝菌样品之间存在一定的遗传差异。5个牛肝菌样品的ITS和IGS1序列聚类分析均表明,供试样品被分为两大类,其中样品YX1-2和QJ3-4聚为一个类群,样品CX2-10、QJ3-5和YX1-3聚为另一个大的类群,两种分析手段相互印证,且ITS序列聚类分析能将云南不同地点的牛肝菌鉴定到属甚至种的水平,上述分析结果表明ITS和IGS1序列分析相结合可作为牛肝菌亲缘关系鉴定的有效分子标记方法。     

基于双指标分析法和聚类分析法的白刺红外指纹图谱比较研究

采用双指标序列分析法和聚类分析法对7种不同白刺属植物的红外指纹图谱进行比较分析,利用共有峰率和变异峰率2个指标,以不同来源样品的红外指纹图谱为标准,计算出所测样品间的共有峰率和变异峰率,并按照共有峰率的大小建立不同的双指标序列分析法,研究各产地白刺的异同。结果表明:不同来源的植物样品中G3与G6的成分最为相似,相似度为57.89;G3与G5次之,相似度为52.63;G1、G7与其他各样品的相似程度最低。红外光谱指纹图谱结合聚类分析或双指标序列法,可以快速、无损地鉴别不同产地的白刺,为区别不同产地不同品种白刺提供一种切实可靠的方法。     

四川凉山地区烟草黑胫病菌的ITS序列分析

本研究利用真菌核糖体基因转录间隔区(ITS)通用引物,PCR扩增四川省凉山州不同地区的20株烟草黑胫病菌的ITS序列,并对PCR产物进行了克隆测序。结果表明,20个供试菌株的ITS1-5.8S-ITS2总长均为803 bp;各菌株之间的ITS序列同源性达到99.3%以上;与GenBank报道的寄生疫霉台湾烟草分离株Phytophthora parasitica(GU111675.1)的同源性在99.6%~100%之间;但各菌株ITS序列的个别位点存在突变,这些突变主要集中在ITS2区上,其中第468处的突变具有一定地域性,主要为德昌和西昌的菌株。利用MEGA6软件对20个供试菌株及GenBank中登陆的15个疫霉属菌株序列进行聚类分析,供试菌株与GenBank上登录的GU111675.1、KF010303.1、AJ854295.1、KC768775.1、L41383聚在同一聚类组上,均为Phytophthora parasitica(或Phytophthora nicotianae),其它疫霉种聚在不同的聚类组上,遗传关系较远,这表明来自四川凉山不同地区的20株烟草黑胫病分离菌株均为寄生疫霉(Phytophthora parasitica)。     

基于内转录间隔区序列对云南12株鹅膏菌的分子分析

目的 基于内转录间隔区(internal transcribed spacer, ITS)序列对云南省12株鹅膏菌进行分类鉴定。方法 采用十六烷基三甲基溴化铵法(cetyltrimethylammonium Ammonium Bromide, CTAB)法提取12株鹅膏菌DNA, 以ITS4和ITS5为引物进行PCR扩增, 完成DNA序列测序, 对序列进行分析并对发育树进行构建。 结果 12个样品的ITS序列长度574~755 bp, GC含量39%~47%, 平均遗传距离为0.329, YN05、YN03与其他10株鹅膏亲缘关系较远。结论 ITS序列高度保守, 在真菌的科属种上能够初步实现对物种的鉴定和系统 发育分析, 为建立云南省鹅膏属分子数据库提供基础数据。     

基于双指标分析法和聚类分析法的香椿红外指纹图谱研究

采用双指标序列分析法和聚类分析法对13个不同产地的香椿样品(T1~T13)的红外指纹图谱进行比较分析。结果表明:不同产地香椿的红外光谱基本一致,主要显示为多糖、蛋白质、氨基酸和多酚的特征峰,但在3400~2800和1700~1000 cm-1波段范围吸收峰的数目、形状和强度存在一定差异。不同产地香椿样品之间的共有峰率在27.78%~84.62%,变异峰率在0~160%。产地相近的T7和T1、T4之间有较高的共有峰率(均为84.62%)和很低的变异峰率(均为18.18%);产地相距较远的T5和T8、T10、T12之间有很低的共有峰率(≤ 31.25%)和很高的变异峰率(≥ 100%)。聚类分析直观地将不同产地香椿样品分为四类,方法相对简便,但精度相对较低。因此,红外指纹图谱结合双指标序列法或聚类分析,可以作为鉴别不同产地香椿的辅助手段,为香椿产地鉴别提供一定的依据。     

云南省6种鹅膏菌DNA条形码序列分析

目的 对云南省6种鹅膏菌的核糖体大亚基(nuclear large-subunit ribosomal DNA, nLSU rDNA)及内转录间隔区(internal transcribed spacer, ITS)DNA序列进行比对分析, 探究其亲缘关系以找到合适的鉴定鹅膏菌DNA条形码序列的方法。方法 采用试剂盒离心柱法提取6种鹅膏菌DNA, 以ITS4/ITS5作为ITS序列引物和LR5/LROR作为nLSU序列的引物进行PCR扩增, 后送至华大基因科技有限公司进行测序。所返回的序列进行遗传距离分析及邻接树构建。结果 球基鹅膏、红托鹅膏与小豹斑鹅膏3种鹅膏菌亲缘关系较近。以ITS序列分析, 三者遗传距离在0.0202~0.0801之间。以nLSU序列分析, 三者遗传距离在0.0031~0.0303之间。黄毒蝇鹅膏与赭盖鹅膏菌H2在ITS序列中与黄毒蝇鹅膏20、21、H7、D1、D16、D24亲缘关系较近, 遗传距离在0.0462~0.0479。赭盖鹅膏菌7与黄毒蝇鹅膏D14两株菌遗传距离仅有0.1528。从邻接树来看ITS序列存在种内多, 聚类分析结果不稳定, 明显不同的2个种聚为一支。而nLSU序列可将各种分离开。结论 建议鹅膏菌快速鉴定选用nLSU序列作为主要鉴定序列, ITS序列作为辅助序列。     

DNA条形码技术在淀粉掺假鉴别中的应用

基于植物DNA条形码技术建立淀粉及其加工制品成分鉴别检测方法。通过提取淀粉5大物种苜蓿、马铃薯、木薯、番薯和玉米的基因组DNA为模板,分别以ITS2、matK、rbcL和trnH-psbA基因通用引物进行聚合酶链式反应扩增,并将测序结果提交GenBank数据库BLAST比对,评价不同植物DNA条形码序列对其鉴别能力。筛选出ITS2＋trnH-psbA为较适组合序列,并对自行采购的5 个淀粉样品和4 个粉条样品进行检测。     

三七中高产人参皂苷Rd真菌的鉴定

从云南文山三七根茎上分离得到具有转化人参皂苷活性的真菌。采用的方法是:利用平板划线分离法,从三七根茎中分离纯化得到2株真菌,用该菌株对三七进行液态发酵。通过HPLC检测,对发酵前后皂苷变化进行分析,对两株真菌进行形态显微鉴定及ITS序列分析。结果表明,菌株形态及显微特征和产孢结构与木霉属一致,利用ITS序列与相关类群构建聚类树,确定实验2菌株ITS总长度为550bp,与长枝木霉Trichodermalongibrachiatum Rifai株菌(ATCC18648T、ATCC208859、DAOM167674、DAOM231259)仅在全ITS第134位点上发生间或缺失突变,鉴定2株真菌均为长枝木霉T.longibrachiatum。HPLC分析显示,菌株能够将三七中量多的大极性皂苷转化为小极性皂苷,并高产人参皂苷Rd。结论由此可以证明长枝木霉具有转化三七中皂苷的活性。     

Administration of internal teat sealant in primigravid dairy heifers at different times of gestation to prevent intramammary infections at calving

Intramammary infections (IMI) are common in primigravid dairy heifers and can negatively affect future milk production. Bismuth subnitrate-based internal teat sealants (ITS) have been used to prevent prepartum IMI in dairy heifers by creating a physical barrier within the teat, preventing pathogens from entering the gland, though determination of when to administer ITS in heifers has yet to be investigated. The objectives of this study were to determine if administration of ITS in primigravid heifers reduced the odds of IMI at calving and if administration of ITS at different stages of gestation (75 vs. 35 d prepartum) affected the odds of IMI at calving. A total of 270 heifers were used at a single farm. One quarter of each heifer was randomly chosen to be aseptically sampled and administered ITS 75 d prepartum (ITS75), another quarter of each heifer was sampled and received ITS 35 d prepartum (ITS35), whereas the remaining 2 quarters of each heifer served as control quarters (CON) and were not sampled before calving. Within 12 h of calving, aseptic colostrum samples were collected from all quarters to determine quarter infection status. When an IMI was caused by mastitis pathogens other than non-aureus staphylococci (NAS), CON quarters were 3 times [95% confidence interval (CI): 1.4–6.3] and 2.5 times (95% CI: 1.2–4.9) more likely to be infected at calving than ITS75 and ITS35 quarters, respectively. For IMI with NAS, CON quarters were 5.8 (95% CI: 3.2–10.5) and 6.4 (95% CI: 3.4–12.0) times more likely to be infected than ITS75 and ITS35 quarters, respectively. Odds of IMI at calving was similar between ITS75 and ITS35 quarters for both NAS (odds ratio = 0.9) and other pathogens (odds ratio = 1.2). Results indicate that ITS administration at either 75 and 35 d prepartum reduced IMI prevalence at calving in primigravid dairy heifers. Farm specific factors may influence prevalence and timing of heifer IMI and earlier administration of ITS provides an extended period of protection for the developing gland.     

Using ITS2 PCR-RFLP to generate molecular markers for authentication of Sophora flavescens Ait

BACKGROUND: Dried root of Sophora flavescens Ait. is a medicinal material occasionally misused or adulterated by other species similar in appearance. In this study the internal transcribed spacer (ITS) regions of DNA samples of S. flavescens Ait. collected from different areas of Taiwan were amplified by polymerase chain reaction (PCR) and compared. The effectiveness of using ITS2 PCR restriction fragment length polymorphism (RFLP)‐generated markers to differentiate S. flavescens Ait. from possible adulterants was also evaluated. RESULTS: The S. flavescens Ait. samples collected from different areas were extremely low in ITS sequence variability at species level. ITS2 PCR‐RFLP coupled with restriction enzymes Sac I, Sac II, Xho I or Pvu I produced specific fragments for all tested variants. ITS2 PCR‐RFLP coupled with Sac II was further performed to identify mixtures of DNA extracts of S. flavescens Ait. and Sophora tomentosa L. in various ratios. The developed ITS2 PCR‐RFLP markers could detect mixed DNA samples of S. flavescens Ait./S. tomentosa L. up to a ratio of 10:1. CONCLUSION: The present study demonstrates the usefulness of ITS2 PCR‐RFLP coupled with pre‐selected restriction enzymes for practical and accurate authentication of S. flavescens Ait. The technique is also suitable for analysing S. flavescens Ait. mixed with other adulterants. Copyright © 2011 Society of Chemical Industry     

An observational cohort study on persistency of internal teat sealant residues in milk after calving in dairy cows

Our objectives were to evaluate the prevalence of quarters with an observable internal teat sealant (ITS) plug at first milking following calving and investigate persistency of ITS residues in milk after calving. An observational cohort study was carried out on 557 quarters of 156 cows treated with ITS in 6 farms in Quebec, Canada. The presence of an ITS plug at first milking and ITS residues in milk at each milking were observed by producers. The effects of various factors on the odds of observing an ITS plug and persistency of ITS residues in milk were studied using generalized logistic mixed and generalized negative binomial mixed models, respectively. Milk samples were taken on the day before dry-off and on 2 occasions after calving for bacterial identification to detect intramammary infection (IMI) using bacteriological culture followed by MALDI-TOF identification. The association between the absence of an ITS plug and the presence of new IMI was assessed using a mixed logistic regression model. Internal teat sealant plugs after calving were more often observed in rear quarters and in quarters receiving ITS alone at drying-off versus antimicrobial and ITS. We observed an average (standard deviation) persistency of 4.0 d (2.3 d). When an ITS plug was still present at first milking (83% of quarters), the elimination of ITS residues in milk after calving was significantly longer (4.5 d, on average) compared with 1.2 d when an ITS plug was absent. In cows with an ITS plug at calving, we observed a higher number of days of excretion in older cows. When a plug could not be observed, rear quarters, older cows, and cows with a long dry period duration excreted ITS residues for a significantly longer period. The lack of a significant association between the absence of a plug and the odds of new IMI at calving suggests that despite the loss of the plug, cows were still protected against new IMI. Although we were able to highlight some statistically significant risk factors explaining persistency of ITS residues following calving, observed differences were often relatively small and, perhaps, not clinically relevant. In conclusion, an ITS plug was present until first milking after calving for 83% quarters, quarters without an ITS plug at first milking appeared to have been protected from new IMI, and ITS residues could be observed in milk up to 12 d in milk.     

PCR detection of Alternaria spp. in processed foods, based on the internal transcribed spacer genetic marker

The genus Alternaria is considered one of the most important fungal contaminants of vegetables, fruits, and cereals, producing several mycotoxins that can withstand food processing methods. Conventional methods for Alternaria identification and enumeration are laborious and time-consuming, and they might not detect toxigenic molds inactivated by food processing. In this study, a PCR method has been developed for the rapid identification of Alternaria spp. DNA in foodstuffs, based on oligonucleotide primers targeting the internal transcribed spacer (ITS) 1 and ITS2 regions of the rRNA gene. The specificity of the Alternaria-specific primer pair designed (Dir1ITSAlt-Inv1ITSAlt) was verified by PCR analysis of DNA from various Alternaria spp., and also from several fungal, bacterial, yeast, animal, and plant species. The detection limit of the method was 10(2) CFU/ml in viable culture, heated culture, or experimentally inoculated tomato pulp. The applicability of the method for detection of Alternaria spp. DNA in foodstuffs was assessed by testing several commercial samples. Alternaria DNA was detected in 100% of spoiled tomato samples, 8% of tomato products, and 36.4% of cereal-based infant food samples analyzed.     

Urine cotinine as an index of smoking status in smokers during 96-hr abstinence: comparison between gas chromatography/mass spectrometry and immunoassay test strips.

Biomarkers such as carbon monoxide (CO) and cotinine (a nicotine metabolite) are used in tobacco cessation studies to assess smoking status. CO is easy to assess, is inexpensive, and provides immediate results. However, the short half-life of CO may limit its ability to identify smokers who have abstained for several hours. Quantitative methods (e.g., gas chromatography/mass spectrometry, or GC/MS) for measuring urine cotinine, which has a longer half-life, are valid and reliable, though costly and time consuming. Recently developed semiquantitative urine cotinine measurement techniques (i.e., urine immunoassay test strips, or ITS) address these disadvantages, though the value of ITS as a means of identifying abstaining smokers has not been evaluated. The present study examined ITS as a measure of smoking status in temporarily abstaining smokers. A total of 236 breath and urine samples were collected from smokers who participated in two separate studies involving three independent, 96-hr (i.e., Monday-Friday), Latin-square-ordered, abstinence or smoking conditions; a minimum 72-hr washout separated each condition. Each urine sample was analyzed with GC/MS and ITS. Under these study conditions, CO demonstrated moderate sensitivity (83.1%) and strong specificity (100%), whereas ITS assessment showed strong sensitivity (98.5%) and weak specificity (58.5%). In this study of short-term abstinence, ITS classified as nonabstinent nearly half of the samples collected from abstaining smokers. However, it classified nearly all nonabstinent smokers as currently smoking. Validation of ITS using GC/MS results from smokers undergoing more than 96 hr of abstinence may be valuable.     

Internal transcribed spacer (ITS) sequence-based characterization of fungal isolates from multiple yogurt facilities—A case study

Fungal spoilage remains a significant issue in dairy product quality, especially for cultured dairy products such as yogurt formulated without preservatives such as potassium sorbate. Fungal contamination can occur throughout the processing continuum, from the dairy farm environment to the finished product processing environment. As molecular characterization of fungal isolates is used more frequently, we obtained fungal isolates obtained in 2 yogurt processing facilities as part of routine fungal testing of raw materials (e.g., fruit preparations, added ingredients), in-process product samples, environmental samples (e.g., air plates, equipment surfaces such as valves, face plates, air nozzles), and finished product samples, to determine whether internal transcribed spacer (ITS) barcoding data would be helpful to support source tracking of fungal contamination issues. Internal transcribed spacer PCR amplification and sequencing allowed us to classify the 852 isolates from these 2 facilities into 200 unique ITS allelic types (AT), representing the phyla Ascomycota (743 isolates), Basidiomycota (97 isolates), and Mucoromycota (12 isolates). Thirty ITS AT were isolated from both facilities; 62 and 108 ITS AT were isolated from only facility A or only facility B, respectively. Nine ITS AT were each represented by more than 20 isolates; these AT comprised 53% of the 852 isolates. The considerable diversity of fungal isolates even within a single facility illustrates the challenge associated with controlling fungal contamination of dairy products. The ITS barcoding technique, however, did show promise for facilitating the source tracking of fungal contamination, particularly for ITS AT over-represented in a given facility. For example, we found evidence for equipment-specific reservoirs for 2 AT (14 and 219) in facility B. Our data suggest that despite its limited discriminatory power, ITS sequencing can provide initial information that can help trace fungal contamination along the processing continuum. However, development and implementation of discriminatory subtyping methods will be needed to further improve the ability to identify sources of fungal contamination in dairy facilities. Developing and implementing sampling plans that comprehensively capture yeast and mold diversity in a given processing facility remain a considerable challenge.     

ITS序列在酵母批量分子鉴定中的适用性

内部转录间隔区(internal transcribed spacer,ITS)序列是目前最常用的酵母分子鉴定标识之一。该文利用ITS序列同源性分析法对保藏于中国高校工业微生物资源与信息中心的623株酵母分离物进行了鉴定。实验结果显示:581株酵母分离物(93.3%)可通过ITS序列直接鉴定到种一级,40株分离物(6.4%)可鉴定至属一级,仅有2株酵母分离物无法通过ITS序列获得有效鉴定。上述结果表明,ITS序列在大多数酵母种属鉴定中具有很高的敏感性和特异性,可以作为第一分子标识用于大批量酵母分离物的分子鉴定。