毛囊蠕形螨与皮脂蠕形螨基因组DNA的RAPD分析和序列比对

【目的】分析毛囊蠕形螨Demodex folliculorum（D.f.）和皮脂蠕形螨D. brevis（D.b.）基因组DNA的多态性, 对相关条带进行测序分析。【方法】采用改良小昆虫DNA提取法提取两种人体蠕形螨基因组DNA, 选择RAPD技术对其进行多态性分析, 将相关条带分别与pMD18-T载体连接, 克隆、测序后进行酶切鉴定和分析。【结果】毛囊蠕形螨共扩增15条带, 皮脂蠕形螨共扩增12条带;两种蠕形螨既有共有条带, 又有特异性条带;根据条带差异计算得到两种间的遗传距离为0.5556. 毛囊蠕形螨约800 bp处特异性条带测序结果显示, 序列片段长度为855 bp（GenBank登录号为FI277970）;特异性引物扩增和酶切鉴定均为毛囊蠕形螨所特有. 序列比对显示与阿糖胞苷DNA区域结合蛋白有46%的序列相似度。两种人体蠕形螨约300 bp处共有条带序列分析显示, 碱基序列均为341 bp（GenBank登录号分别为D.f. FI520176;D.b. FI520175）, 在第84和第165位点有2个碱基不同, 分别是A/G和C/T互换, 同源性高达99.4%. 但未发现有开放阅读框和相似度高的序列。 【结论】序列片段为855 bp的特异性条带为毛囊蠕形螨所特有;341 bp碱基序列为毛囊蠕形螨和皮脂蠕形螨所共有, 同源性高达99.4%. RAPD技术可用于两种人体蠕形螨基因组DNA的多态性分析和物种鉴定。     

几种环境因素对两种人体蠕形螨生活力的影响

本文就寄生人体的毛囊蠕形螨和皮脂蠕形螨在实验室条件下,对其在各种温度、湿度和酸碱度中的存活时间进行了观察。 在36℃,高湿的环境中,毛囊蠕形螨和皮脂蠕形螨成虫的存活时间分别达94和95小时。高温与干燥对其生存不利。两种蠕形螨对酸性环境的耐受力都强于碱性环境,特别皮脂蠕形螨更为突出。     

薄荷油体外抗蠕形螨效果及杀螨机制

采用透明胶带粘贴过夜法获取2种人体蠕形螨,随机分组,观察不同浓度的薄荷油的杀虫效果,并在MoticDMB5图像采集软件系统下拍摄虫体在薄荷油作用下的虫体死亡过程。结果表明,薄荷油有很强的体外杀灭2种人体蠕形螨的作用,尤其对皮脂蠕形螨的杀灭作用显著。随着药物浓度的增加及作用时间的延长,蠕形螨死亡率增高。12.5%、3.125%分别是薄荷油体外杀灭毛囊蠕形螨和皮脂蠕形螨的最适杀螨浓度。薄荷油作用于蠕形螨,可见虫体收缩扭动,活动加剧,消化管剧烈收缩,毛囊蠕形螨和皮脂蠕形螨体壁均有渗出物外溢。虫体表现为兴奋—痉挛—失水—松弛—死亡的典型症状。薄荷油对人体蠕形螨的杀灭机制主要通过神经毒性和直接毒杀作用,造成虫体破裂脱水而死亡。薄荷油是一种极具开发潜能的高效的天然杀螨药。     

人体蠕形螨爬行的观察

蠕形螨为一永久性寄生螨类,寄生于人体及家畜、哺乳类动物,种类繁多,目前报道的有120余种,常见的有40余种,寄生人体的有毛囊蠕形螨(Demodex folliculorum)、皮脂蠕形螨(Demodex brevis)、毛囊蠕形螨中华亚种(Demodex folliculorum sinensis)等,分别寄生于毛囊及皮脂腺内。可引起酒渣鼻、痤疮、眼睑炎等皮肤病,在人群中感染甚为普遍,同时也证实了其感染途径是通过直接接触而传播。为此,对蠕形螨活动力的研究是进一步探索其传病机制及防治的重要环节。人体蠕形螨在不同温度、湿度及酸碱度条件下生活能力的观察曾有报道(Spickett,1961;陈国定,1985)。但对其爬行规律及速度至今尚未见报道,为此我们对蠕形螨在不同温度条件下的爬行规律及速度作了系统的观察。今将结果报道如下。     

蠕形螨的检查方法

蠕形螨的检查方法蠕形螨（Ｄｅｍｏｄｅｘ）是一类微小的寄生性螨,其成虫呈蠕虫状,雌雄异体。根据曲魁遵所述,此虫大小的２５０μｍ×４０μｍ,虫体可分为头胸部及腹部两部分。该螨寄生于人体和其他哺乳动物皮内的皮脂腺和毛囊管内。寄生于人体的蠕形螨有毛囊形螨（Ｄ...     

动物蠕形螨活动情况初步观察

蠕形螨属Demodex是一类小型螨,寄生于人、家畜、野生动物的皮肤毛囊和皮脂腺内,不仅影响人类健康,并能引起家畜和珍稀动物的蠕形螨病,严重时可以导致动物死亡,目前尚无理想的特效药物。近年来,国内外学者对蠕形螨的形态结构,致病力,感染途径以及诊疗方法等作了不少研究,但对动物蠕形螨的活动情     

人体蠕形螨寄生率与工种关系的初步调查

目的：探讨人体蠕形螨寄生率与工种的关系;方法：采用手指挤压和刮取法刮取皮脂分泌物在显微镜下检查;结果：不同工种的人群蠕形螨寄生率有显著差异（χ２＝８１３２,ｆ＝４,Ｐ＜００５）;结论：工作环境和个人卫生条件决定蠕形螨寄生率的高低。     

大熊猫蠕形螨病组织病变的初步观察

大熊猫蠕形螨病的临床症状和危害已有记述〔1,2〕。本文通过一例病猫皮损活组织块,经石蜡包埋做成连续切片,HE染色,厚度为8μm,镜下见到表皮角质层增厚,棘层稍增厚,基层正常。毛囊漏斗部与皮脂腺导管都有不同程度的扩大,其内毛根脱落较多。同时,被许多蠕形螨碎片及完整的蠕形螨充塞。部分毛囊的上皮细胞有坏死脱落、核溶解及核碎裂现象;有的含虫毛囊周围的间质内有大量嗜中性粒细胞及嗜酸性粒细胞浸润;个别毛囊腔内充满大量嗜中性粒细胞及脓球;少数汗腺管内及其它结缔组织内也有炎性细胞浸润。组织内大熊猫蠕形螨主要寄生于毛囊上段,即漏斗…     

毛囊蠕形螨的发育形态观察和存活适温范围研究

采用挤压涂片法或透明胶纸粘贴法获取毛囊蠕形螨,在数码显微镜Motic DM-B5软件系统下进行动态观察、拍摄和测量;然后对离体蠕形螨按批随机分组,观察不同温度下虫体的存活时间和活动力。结果系统地展示了毛囊蠕形螨发育过程各个时期的动态照片,发现毛囊蠕形螨的前若虫期有足4对,若虫期具有活动能力。离体实验表明,毛囊蠕形螨耐低温而不耐高温;存活适温范围在8～30℃之间,发育适温范围在20～30℃之间,发育最适温度为25～26℃;0℃以下或37℃以上温度对蠕形螨生存不利,54℃为致死温度,58℃为有效灭螨温度。这一研究结果为蠕形螨的体外培养和防治工作提供了可靠的理论依据。     

人体正常皮肤蠕形螨生态分布的研究

本文报道了大连地区人体正常皮肤蠕形螨的生态分布。共检查551人,阳性153人,定居率为27.8％,检出毛囊螨和皮脂螨共两种。用透明胶带定量计数检查法,研究蠕形螨在面部的分布,结果表明蠕形螨主要分布于颏、鼻、颊和额部位。对10例阳性者,在面部检出毛囊螨和皮脂螨共85只,其中颏31、鼻26、颊17、额11只螨,总定居度为每条胶带(6cm~2)2.1只螨,定居度以颏部位为最高,鼻、颊、额次之。     

Complete sequence analysis of 18S rDNA based on genomic DNA extraction from individual Demodex mites (Acari: Demodicidae)

The study for the first time attempted to accomplish 18S ribosomal DNA (rDNA) complete sequence amplification and analysis for three Demodex species (Demodex folliculorum, Demodex brevis and Demodex canis) based on gDNA extraction from individual mites. The mites were treated by DNA Release Additive and Hot Start II DNA Polymerase so as to promote mite disruption and increase PCR specificity. Determination of D. folliculorum gDNA showed that the gDNA yield reached the highest at 1 mite, tending to descend with the increase of mite number. The individual mite gDNA was successfully used for 18S rDNA fragment (about 900 bp) amplification examination. The alignments of 18S rDNA complete sequences of individual mite samples and those of pooled mite samples ( ≥ 1000mites/sample) showed over 97% identities for each species, indicating that the gDNA extracted from a single individual mite was as satisfactory as that from pooled mites for PCR amplification. Further pairwise sequence analyses showed that average divergence, genetic distance, transition/transversion or phylogenetic tree could not effectively identify the three Demodex species, largely due to the differentiation in the D. canis isolates. It can be concluded that the individual Demodex mite gDNA can satisfy the molecular study of Demodex. 18S rDNA complete sequence is suitable for interfamily identification in Cheyletoidea, but whether it is suitable for intrafamily identification cannot be confirmed until the ascertainment of the types of Demodex mites parasitizing in dogs.     

Demodex spp. in the hair follicles of rhesus macaques (Macaca mulatta)

The perineal or perineal and facial skin were evaluated on 53 rhesus macaques as part of a necropsy protocol. Microscopic evaluation of H & E stained skin sections revealed 19 animals positive for Demodex spp. Mites were seen within all portions of the hair follicles. Infestation varied from minimal to severe. Mites were found in macaques of all ages and in both sexes. Reaction to the mites ranged from no reaction, to minimal follicular epidermal hyperplasia to furunculosis. Immune status of the animal did not determine infestation but immune compromised macaques had more severe lesions. This is the first known report of Demodex spp. in rhesus macaques.     

Risk factors and prevalence of Demodex mites in young adults

Demodex mites are ectoparasites often found in follicles of facial skin. Their role in human diseases is under investigation, and a growing number of studies indicated that they contribute to chronic inflammatory conditions of the skin, such as rosacea, blepharitis, otitis externa, alopecia and folliculitis. In our study we tested 96 healthy adults for the presence of Demodex mites. Risk factors influencing presence of mites and skin types of the tested individuals were evaluated. We found Demodex folliculorum or Demodex brevis in 17.7% of the samples, more frequently in males (21.9%) and in older adults (20%). Use of make-up seems to reduce the likelihood of Demodex carriage, while pet ownership, use of shared items and living in close contact with older adults had no significant influence of presence of mites. Demodex positive individuals described their skin to be drier, more prone to erythema, but less for folliculitis compared to Demodex negative subjects.     

On-line cell lysis of bacteria and its spores using a microfluidic biochip

Optimal detection of pathogens by molecular methods in water samples depends on the ability to extract DNA rapidly and efficiently. In this study, an innovative method was developed using a microfluidic biochip, produced by microelectrochemical system technology, and capable of performing online cell lysis and DNA extraction during a continuous flow process. On-chip cell lysis based on chemical/physical methods was performed by employing a sufficient blend of water with the lysing buffer. The efficiency of lysis with microfluidic biochip was compared with thermal lysis in Eppendorf tubes and with two commercial DNA extraction kits: Power Water DNA isolation kit and ForensicGEM Saliva isolation kit in parallel tests. Two lysing buffers containing 1% Triton X-100 or 5% Chelex were assessed for their lysis effectiveness on a microfluidic biochip. SYBR Green real-time PCR analysis revealed that cell lysis on a microfluidic biochip using 5% Chelex buffer provided better or comparable recovery of DNA than commercial isolation kits. The system yielded better results for Gram-positive bacteria than for Gram-negative bacteria and spores of Gram-positive bacteria, within the limits of detection at 10³ CFU/ml. During the continuous flow process in the system, rapid cells lysis with PCR-amplifiable genomic DNA were achieved within 20 minutes.     

临床标本细菌基因组DNA提取方法探讨

目的优化细菌基因组DNA提取方法,使其适合临床细菌分子生物学检测需要。方法分别采用专用DNA提取液法、热裂解法、溶菌酶法、热裂解法与碱性裂解法组合改良法,对纯培养细菌和临床标本中细菌基因组DNA进行提取。结果专用DNA提取液法、溶菌酶法提取成功率为100%,热裂解法革兰阳性菌提取成功率为0%,革兰阴性菌成功率为100%,碱性裂解液法在NaOH浓度大于4 mmol时提取成功,临床标本在NaOH溶液超过20 mmol/L并含2%SDS时细菌基因组DNA的提取成功率为100%。结论热裂解法与碱性裂解法组合改良法提取细菌基因组DNA方便快速、简单实用,适用临床标本检测。