Y chromosome detection by Real Time PCR and pyrophosphorolysis-activated polymerisation using free fetal DNA isolated from maternal plasma

Objectives To validate the use of Real Time PCR, a widely used technique that can detect very low levels of Y chromosomal sequence, and to assess the use of a highly sensitive PCR technique, pyrophosphorolysis-activated polymerisation (PAP), for fetal sex determination using free fetal DNA (ffDNA). Methods The fetal sex was determined by Real Time PCR in 58 pregnancies using ffDNA isolated from maternal plasma. In parallel with the Real Time PCR experiments, the presence of Y chromosome sequence was also determined using PAP on 54 isolated ffDNA samples. Results Both techniques detected Y chromosome sequence at very low levels with 98% specificity and 100% sensitivity (Real Time n = 44, PAP n = 54). Furthermore, the PAP technique was shown to be more robust than the Real Time PCR as none of the samples tested failed to meet the acceptance criteria. Combining the two techniques for male fetal sex detection from maternal blood plasma increases the sensitivity and specificity to 100% in this series. Conclusions This study shows that both Real Time PCR and PAP can be used for Y chromosome detection on ffDNA. Furthermore, by using PAP in combination with Real Time PCR more reliable early prenatal sexing can be performed using ffDNA. Copyright © 2007 John Wiley & Sons, Ltd.     

Morphological and cytogenetic analysis of intact oocytes and blocked zygotes

We examined cytological and cytogenetic parameters of 1076 oocytes and 385 zygotes that failed to develop post in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Out of 1076 oocytes, 894 (83%) arrested oocytes showed a first polar body and were thus assumed arrested at metaphase II while the remainder showed no polar body. In the group of oocytes with a polar body, 20.5% had an abnormal karyotype. Cytologically, premature sperm chromosome condensation was noted in 28.3% of uncleaved oocytes. This high PCC can be explained by the different grades of oocyte maturity from one center to another. Oocytes from older women showed no increased aneuploidy but did show increased premature chromosome condensation. Analysis by classical technique of 220 uncleaved zygotes showed 91 with highly condensed chromosomes, 53 with asynchrony of condensation, 31 with pulverized chromosomes, and 45 arrested at the first somatic metaphase. Out of 385 arrested zygotes, 165 were explored by in situ hybridization. FISH using a set of 7 chromosome-specific probes showed aneuploidy in the chromosomes analyzed (13, 16, 18, 21, 22, X, Y) in 21.8% of blocked zygotes (19–25% depending on morphology). Extrapolating to other chromosomes, we expect that a vast majority of blocked zygotes and oocytes probably carry chromosome abnormalities. These data demonstrate the contributions of chromosome disorder in early embryo development blocking and implantation failure. Certainly, the issue of cytoplasm and nuclear immaturity and their relation to each other and to chromosome abnormalities provides a fertile area for future investigation in ART. Copyright © 2003 John Wiley & Sons, Ltd.     

阴离子粘土(LDH)对DNA吸附行为的研究

以4种阴离子粘土做吸附剂,研究了阴离子粘土对DNA的吸附行为.同时,采用XRD、FTIR、UV-vis等表征手段对吸附前后的材料进行研究.吸附结果显示,二元阴离子粘土对DNA的吸附量高于三元阴离子粘土;3:1型阴离子粘土对DNA吸附力强于2:1型阴离子粘土.4种材料对DNA的吸附均符合Langmuir、Freundlich两种吸附等温模型,且Langmuir吸附等温模型拟合度更高,说明阴离子粘土对DNA的吸附为单层吸附.XRD结果显示,吸附前后阴离子粘土基本结构并未发生改变,晶形完好,层间距未有明显变化,表明阴离子粘土对DNA的吸附仅发生在表面,DNA并未进入阴离子粘土层间结构中.UV-vis及电泳结果显示,吸附前后DNA的构型并未发生改变,阴离子粘土的吸附并未对DNA产生较大的影响.     

长江江豚监测现状及展望

长江江豚(Neophocaena asiaeorientalis)是唯一淡水生活的鼠海豚类,近年来种群数量严重下降,2013年被世界自然保护联盟红色名录列为“极度濒危”,2021年升级为我国国家一级重点保护野生动物,2022年种群有所恢复。长江江豚是长江生态系统健康的指示物种,我国科学家自20世纪50年代开始监测至今,监测方法不断更新完善,对长江江豚现状的了解也越来越充分。该文回顾了截线抽样法、水下被动声学及自动实时监测系统、无人机、环境DNA等监测方法的应用及取得的成效,分析监测方法存在的不足,预测技术发展趋势,提出改进建议,为长江江豚的监测及保护提供基本参考。     

Use of genetic,climatic, and microbiological data to inform reintroduction of a regionally extinct butterfly

Species reintroductions are increasingly used as means of mitigating biodiversity loss. Besides habitat quality at the site targeted for reintroduction, the choice of source population can be critical for success. The butterfly Melanargia russiae (Esper´s marbled white) was extirpated from Hungary over 100 years ago, and a reintroduction program has recently been approved. We used museum specimens of this butterfly, mitochondrial DNA data (mtDNA), endosymbiont screening, and climatic‐similarity analyses to determine which extant populations should be used for its reintroduction. The species displayed 2 main mtDNA lineages across its range: 1 restricted to Iberia and southern France (Iberian lineage) and another found throughout the rest of its range (Eurasian lineage). These 2 lineages possessed highly divergent wsp alleles of the bacterial endosymbiont Wolbachia. The century‐old Hungarian specimens represented an endemic haplotype belonging to the Eurasian lineage, differing by one mutation from the Balkan and eastern European populations. The Hungarian populations of M. russiae occurred in areas with a colder and drier climate relative to most sites with extant known populations. Our results suggest the populations used for reintroduction to Hungary should belong to the Eurasian lineage, preferably from eastern Ukraine (genetically close and living in areas with the highest climatic similarity). Materials stored in museum collections can provide unique opportunities to document historical genetic diversity and help direct conservation.     

基于环境DNA宏条形码的青岚湖自然保护区鱼类组成及分布特征

为探究在不同引物、不同参考数据库下环境DNA技术检测结果的差异,于2021年4月,采用环境DNA宏条形码技术分析了青岚湖鱼类多样性。选取16S rRNA及Cytb 2种引物,NCBI及本地数据库2种数据库,分别进行比对注释。结果表明:在青岚湖29个采样点中,共检测到7目15科43属64种鱼类,其中在16S-NCBI情形下获得4目9科19属20种鱼类,在16S-本地数据库情形下获得6目13科27属38种鱼类,在Cytb-NCBI情形下获得4目6科16属19种鱼类,在Cytb-本地数据库情形下获得2目5科15属20种鱼类。在青岚湖29个采样点中,鱼类更多地分布于湖面宽阔的中间地带(如S31附近),且南部较北部更少,鱼种的分布呈现一定的空间相似性。就研究区域而言,选择16S rRNA引物及本地数据库可以获得更全面的鱼类物种。通过与青岚湖鱼类历史数据比对发现,环境DNA技术在研究区域具有较强的适用性,如能选择适当的引物和本地数据库,可以更全面地反映研究区域的物种组成情况。     

基于环境DNA的长江中华鲟分布特征探究

中华鲟（Acipenser sinensis）是我国长江流域的旗舰物种,在长江十年禁渔的大背景下,研究中华鲟无损化检测技术具有重要意义。环境DNA（eDNA）技术是一种环境友好型生物监测技术,可以在不直接观察或捕获生物体的情况下对物种进行检测。从文献中筛选出可以用于检测中华鲟eDNA的特异性引物,于2020年9月在长江中下游选取4个中华鲟常出现的区域,进行各断面立体式采样;提取16个点位的eDNA,使用筛选得到的引物对中华鲟进行eDNA的检测,以探究中华鲟的分布特征。结果显示,成功筛选到1组可以检测中华鲟eDNA的引物,使用该引物成功检测到包括中华鲟在内的长江4种鲟类的eDNA,共计测得约300万条鲟类序列。依据测序结果分析不同断面检测到的中华鲟eDNA的差异,发现宜昌江段断面的中华鲟eDNA最多,洞庭湖口断面最少,且表层和底层水体的中华鲟eDNA检出也有显著差异。筛选得到的引物可以用于中华鲟eDNA的检测,中华鲟eDNA的检测结果与中华鲟的历史调查和洄游特征较为吻合。不同水深条件中华鲟eDNA的检出量有显著差异,表明在今后的调查中采用混合或者立体采样可以更加全面地进行中华鲟eDNA的检测。