Non‐random expression of ribosomal DNA units in a grasshopper showing high intragenomic variation for the ITS2 region

We analyse intragenomic variation of the ITS2 internal transcribed spacer of ribosomal DNA (rDNA) in the grasshopper Eyprepocnemis plorans, by means of tagged PCR 454 amplicon sequencing performed on both genomic DNA (gDNA) and RNA‐derived complementary DNA (cDNA), using part of the ITS2 flanking coding regions (5.8S and 28S rDNA) as an internal control for sequencing errors. Six different ITS2 haplotypes (i.e. variants for at least one nucleotide in the complete ITS2 sequence) were found in a single population, one of them (Hap4) being specific to a supernumerary (B) chromosome. The analysis of both gDNA and cDNA from the same individuals provided an estimate of the expression efficiency of the different haplotypes. We found random expression (i.e. about similar recovery in gDNA and cDNA) for three haplotypes (Hap1, Hap2 and Hap5), but significant underexpression for three others (Hap3, Hap4 and Hap6). Hap4 was the most extremely underexpressed and, remarkably, it showed the lowest sequence conservation for the flanking 5.8‐28S coding regions in the gDNA reads but the highest conservation (100%) in the cDNA ones, suggesting the preferential expression of mutation‐free rDNA units carrying this ITS2 haplotype. These results indicate that the ITS2 region of rDNA is far from complete homogenization in this species, and that the different rDNA units are not expressed at random, with some of them being severely downregulated.     

The effect of intratympanic steroid injection frequency in idiopathic sudden sensorineural hearing loss

Purpose

To evaluate the effect of intratympanic steroid injection frequency on hearing outcomes for patients with idiopathic sudden sensorineural hearing loss.

新蚤属9种新蚤核糖体DNA ITS2序列变化

本研究分析了新蚤属 9个种 (Neopsyllabidentatiformis,N abagaitui,N mana ,N pleskei,N siboi,N teratura ,N hongyangensis ,N specialis和N paranoma)的核糖体DNAITS2的序列变化 ,其中二齿新蚤包括两个地理种群。结果表明 ,该序列在所研究的种内相对稳定 ,在种间 ,除N abagaitui,N specialis和N paranoma间有较多的变异外 ,其他种间的变异较少甚至没有。种间的碱基替换率为 0～ 2 78%。根据碱基替换速率推算的新蚤属两次分化时间分别为 3 5～ 10百万年和 1百万年以后。结合分子和形态特征 ,可以认为N hongyangensis是N bidentatiformis的姐妹种。根据该序列得到的系统发育关系树和根据线粒体DNA16sRNA基因所得的系统发育关系树基本一致。而N siboi则出现了线粒体基因组和核基因组进化的不一致性 ,说明该种的起源值得进一步研究     

三种库蚊核糖体DNA第二内转录间隔区(ITS2)序列测定与系统进化分析

对广东省野外采集的库蚊属中2个亚属中的3种库蚊,褐尾库蚊Culex fuscanus(Lutzia、路蚊亚属)、二带喙库蚊Cx.bitaeniorhynchus和致倦库蚊Cx.pipiens quinquefasciatus(Culex、库蚊亚属)进行核糖体DNA第二内转录间隔区(ITS2)序列测定,并与GenBank中已知库蚊属中路蚊亚属、库蚊亚属、新库蚊亚属和黑蚊亚属的11种和伊蚊属1种蚊虫的ITS2进行序列分析。结果表明:褐尾库蚊与同亚属的非洲蚊种(Culex tigripes)亲缘关系最近,基因同源性为65.27%;库蚊亚属的二带喙库蚊与伪杂鳞库蚊基因同源性为75.87%;致倦库蚊与尖音库蚊复合组内淡色库蚊的基因同源性97.13%。在所选库蚊中,黑蚊亚属与伊蚊最接近,其次是新库蚊亚属和路蚊亚属的蚊虫。在库蚊属4个亚属中,路蚊亚属与库蚊亚属有更近的亲缘关系,这也印证了库蚊亚属某些幼虫与路蚊亚属幼虫具有相似习性的遗传基础。系统进化关系分析结果与经典分类学分类系统接近一致,但库蚊亚属的进化可能更具多样性。     

泽泻异地交叉种植前后植株样品rDNA-ITS区序列分析

目的从分子水平上揭示生态环境对泽泻“道地性”的影响。方法将建泽泻、川泽泻种子分别交叉在福建建瓯、四川彭山种植1年,时以上4个泽泻植株样品的18S-26S rDNA—ITS片段进行PCK扩增、测序,并运用clustalx1．83对该区序列进行分析。结果在四川栽培的建泽泻与在福建栽培的建泽泻的ITS片段序列在第614位相差1个碱基;而在福建栽培的川泽泻与在四川栽培的川泽泻序列完全一致。结论改变泽泻生态环境1年,其ITS序列发生变异的可能性很小。     

基于DNA条形码技术的维吾尔药材桑葚基原鉴定研究

该研究通过分析多途径来源的新疆桑属植物及药材样本的ITS2,psbA-trnH序列,为药材分子鉴定提供依据。以51个新疆桑属植物及药材为样本,对其ITS2,psbA-trnH序列进行PCR扩增和测序,用MEGA 6.0计算其种内、种间Kimura 2-parameter(K2P)距离,分析变异位点,并构建NJ鉴别树。ITS2序列分析结果显示,桑Morus alba、鞑靼桑M.alba var.tatarica、黑桑M.nigra种内无变异;桑与鞑靼桑种间无变异;桑与黑桑种间存在13个变异位点,种间平均KP2遗传距离为0.04;桑与药材样本间无信息变异位点,NJ鉴别树可将桑及鞑靼桑与黑桑区分。psbA-trnH序列分析结果显示,桑与黑桑种内各有1个变异位点,3种植物种间存在插入/缺失变异,可相互区分;种间变异与药材样本内变异一致。因此,ITS2序列可将来源于桑、鞑靼桑的药材样本与黑桑区分,psbA-trnH序列可将来源于三者的药材样本区分,为维吾尔药材真伪鉴别及市场监管提供依据。     

Mechanisms of Chinese medical formula Fangji Huangqi Decoction as an effective treatment of nephrotic syndrome based on systems pharmacology

Objective: Flower herbs are an important category of traditional Chinese medicinal materials, some of which are used as healthcare tea in folk. However, the increasing adulteration of medicinal herbs is threatening consumer safety. The adulteration of flower herbs and their healthcare tea products in the market were investigated. Methods: A total of 33 flower herb samples from several retail pharmacies in China were randomly collected and 27 flower healthcare tea samples were purchased online. They were identified using ITS2-based Traditional Chinese Medicine Database (TCMD). Additionally, Lonicerae Japonicae Flos and the adulterants were compared in the ITS2 secondary structures. Results: There were one adulterant (Inulae Flos) in flower herb materials and eight adulterants in healthcare tea samples. Inula linariifolia was an adulterate species of Inulae Flos, Robinia pseudoacacia was of Sophorae Flos, and Lonicera macranthoides was of Lonicerae Japonicae Flos. Sophorae Flos and Lonicerae Japonicae Flos were two healthcare tea products with high adulteration rates. Conclusion: The TCMD is powerful tool to identify flower herbs and the adulterants that frequently occurred in the flower herb market, especially online shops.     

中药材DNA条形码技术研究进展

摘 要 药用植物滥用现象带来的众多有害反应已经引起了国内外广泛的关注。药物安全使用问题亟待解决。DNA条形码是一种强有力的分子鉴定技术,它能弥补传统形态学和化学方法鉴定中药材的不足之处。近年,DNA条形码技术给中药材的鉴定带来了新的视野也获得了众多突出性成绩。一些热门的DNA条形码候选序列如ITS, ITS2, psbA-trnH, rbcL, matK已经用于中药材的鉴定。     

Rapid identification of invasive fungal species using sensitive universal primers-based PCR and restriction endonuclease digestions coupled with high-resolution melting analysis

BackgoundConventional diagnosis of invasive fungal disease from blood cultures is often notoriously delayed and inadequately sensitive. We aimed to develop a universal primers-based polymerase chain reaction (PCR) assay and restriction fragment length polymorphisms (RFLP) for rapid identification of invasive fungal disease (IFD).MethodsWe evaluated 16 clinical fungal species using a combination of PCR assays with 3 different restriction endonucleases targeting various internal transcribed spacer (ITS) regions and high resolution melting analysis (HRMA). Serial samples from 75 patients suspected to have IFD were analyzed for clinical verification.ResultsWe have designed a universal PCR capable of amplifying a portion of the 18S rRNA gene of 16 clinically important fungal species. The restriction patterns of most PCR products generated by EcoRI or double digested by ClaI and AvaI were different, except Aspergillus niger and Aspergillus flavus had a similar pattern, and Aspergillus fumigatus and Aspergillus terreus had a similar pattern. All these species had a unique melting curve shape using the HRMA. Both HRMA and universal PCR had adequate sensitivity, and all sixteen reference fungal species can be clearly distinguished by the universal PCR-RFLP-HRMA assay. With a reference library of 176 clinically relevant fungal strains, and 75 clinical samples from patients with suspicious IFD were tested, our assay identified 100% and 61.1% of isolates from the reference library and clinical samples, respectively.ConclusionsUniversal PCR and RFLP coupled with HRMA could be a highly discriminative and useful molecular diagnostic that could enhance the current diagnostic, treatment, and surveillance methods of invasive fungal disease.     

基于ITS条形码的滇鸡血藤及混伪品分子鉴定

目的：基于内转录间隔区（ITS）序列,运用DNA条形码技术对滇鸡血藤药材及其混伪品进行序列比对分析,根据单核苷酸多态性位点构建滇鸡血藤单倍型数据库,建立快速、准确鉴定滇鸡血藤的分子鉴定方法。方法：以滇鸡血藤及其混伪品为材料,利用DNA提取试剂盒,分别提取滇鸡血藤及其混伪品的总DNA,对ITS序列进行聚合酶链式反应扩增及测序分析,使用DNAMAN软件统计其片段长度及变异位点个数,使用MEGA软件对数据进行分析比对,计算Kimura2-parameter遗传距离,并利用邻接法建立系统发育树进行分析。结果：滇鸡血藤ITS序列片段长度为675 bp,共包括19种单倍型,与其主要混伪品的差异位点为212、223、224 bp,系统发育树显示,ITS序列能够区分滇鸡血藤及其混伪品。结论：ITS条形码可以作为区分鉴定滇鸡血藤及其混伪品的分子条形码,包含19个单倍型的单倍型数据库覆盖了滇鸡血藤全部的单倍型。