应用含内参的多重实时荧光RT-PCR方法快速检测登革病毒和基孔肯雅病毒

目的 建立一种登革病毒、基孔肯雅病毒并含人类基因内参检测的多重实时荧光RT-PCR方法,能在同一反应管内同时检测目前发现的所有来源的登革病毒或基孔肯雅病毒。方法 针对登革病毒3′端非编码区和基孔肯雅病毒结构蛋白E2-6K-E1区以及人体各类组织细胞中均能稳定表达的RNAse P基因,设计了3套特异性引物和探针,建立了1套能同时检测登革病毒、基孔肯雅病毒及含有人类基因检测内参的多重实时荧光RT-PCR方法,对其灵敏性和特异性进行了验证,并对临床发热病人标本进行了应用评估。结果 该方法对检测体外转录合成的登革病毒和基孔肯雅病毒RNA的灵敏性可达最低每个反应10～100拷贝,对检测登革1型病毒和基孔肯雅病毒的灵敏性分别可达最低每个反应0.1 TCID₅₀/mL和1 TCID₅₀/mL。用20株登革病毒、4株基孔肯雅病毒和日本脑炎病毒、西尼罗病毒、黄热病毒、盖塔病毒、辛德毕斯病毒各1株进行检测,方法的特异性均为100%。方法应用于189份发热病人血清标本检测,可准确地鉴定出其中登革病毒或基孔肯雅病毒核酸阳性的标本,且所有血清标本均能被内参引物和探针有效地扩增和杂交。结论 本研究建立了一种高灵敏性、高特异性且含人类基因内参检测的登革病毒和基孔肯雅病毒多重实时荧光RT-PCR检测方法,可作为登革热或基孔肯雅热病人早期快速鉴别诊断的有效工具,也可用于蚊媒携带登革病毒或基孔肯雅病毒的高通量快速筛查。     

LAMP技术在动物病毒病原检测中的应用进展

环介导等温扩增技术(LAMP)是一种具有简单、快速、特异性强和扩增效率高的核酸检测技术,目前已在食品、卫生及疾病预防控制检验等领域得到了诸多应用。本文主要介绍了LAMP技术在实验及临床过程中检测肝炎病毒、流感病毒、HIV-1、狂犬病毒、乙脑病毒、冠状病毒、登革病毒等病毒病原感染的应用进展。     

环介导等温扩增技术及其应用

环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术是一种新型的体外核酸扩增技术,在等温条件下,利用一种具有自动链置换活性的Bst DNA聚合酶和一组特异性引物,对靶DNA序列进行快速扩增,1 h左右可合成109～1010拷贝的靶DNA序列.LAMP技术具有简便、快速、扩增效率和特异性高等特点,近年来该技术广泛应用于病原菌、病毒等病原体的快速分子检测.该文详细介绍LAMP的原理、引物设计及其在微生物分子检测方面的应用.     

环介导等温扩增技术及其在病原生物检测中的应用

环介导等温扩增(LAMP)技术是近年来发展起来的一种新型核酸扩增技术,具有操作简便、快速、特异性高、灵敏性高等特点,自2000年开发以来,在短短的十几年内被广泛应用于细菌、病毒和寄生虫等病原生物的检测,在即时检验和基层实验室推广中有重要意义。     

环介导等温扩增技术及其在病原生物检测中的应用北大核心CSCD

环介导等温扩增(LAMP)技术是近年来发展起来的一种新型核酸扩增技术,具有操作简便、快速、特异性高、灵敏性高等特点,自2000年开发以来,在短短的十几年内被广泛应用于细菌、病毒和寄生虫等病原生物的检测,在即时检验和基层实验室推广中有重要意义。     

快速检测日本血吸虫感染性钉螺LAMP试剂盒的建立及应用

目的构建快速检测日本血吸虫感染性钉螺环介导同温DNA扩增（LAMP）试剂盒,用于血吸虫病流行区感染性钉螺调查。方法采用碱裂解法制备钉螺基因组DNA样品,以缩短检测过程中样品DNA制备时间;对LAMP试剂进行组合优化,采用染料显色方法判定LAMP结果,以减少LAMP检测的操作步骤,避免污染,减少假阳性结果产生;建立批量检测钉螺的LAMP方法,以适用于血吸虫病防治现场大规模钉螺筛查与有感染性钉螺地块的调查。将以上优化技术及试剂整合成快速检测日本血吸虫感染性钉螺LAMP试剂盒。结果制备的日本血吸虫感染性钉螺LAMP检测试剂盒可用于血吸虫流行区现场钉螺快速检测,检测时间可由原来的6h缩短为2h,方法的灵敏性与常规LAMP法相似。该试剂盒能检测出感染后1周钉螺,能对现场钉螺进行大批量检测。结论建立的LAMP试剂盒用于日本血吸虫感染性钉螺检测快速、特异、操作简便,适用于日本血吸虫病流行区感染性钉螺调查。     

基于实时荧光环介导等温扩增的HPV16及HPV18检测体系的建立

目的建立用于人乳头瘤病毒HPV16及HPV18快速检测的实时荧光环介导等温扩增技术。方法运用在线LAMP引物设计软件Primer Explorer V5,针对HPV16、18基因保守序列设计扩增引物,在反应前加入核酸染料SYBR GreenⅠ并对LAMP反应体系中内引物、Mg2+、dNTPs进行优化,优化结果通过实时荧光扩增曲线确定;然后对优化的HPV16、18 LAMP反应体系进行特异性、灵敏度检测及临床标本检测。结果建立的实时荧光环介导等温扩增体系检测HPV16及HPV18的特异性为100%,与qPCR方法的符合率为100%。该体系检测HPV16及HPV18质粒的灵敏度为10拷贝/μl。结论建立的实时荧光环介导等温扩增体系检测HPV16、18简便、快速,灵敏度高,可用于HPV感染的早期筛查。     

逆转录环介导等温扩增技术在诺如病毒基因检测中的应用

目的建立逆转录环介导等温扩增技术(RT-LAMP)快速检测诺如病毒的方法。方法采用逆转录环介导等温扩增技术(RT-LAMP)扩增诺如克病毒特异性基因,并与RT-PCR方法比较。结果与RT-PCR技术相比,RT-LAMP法更加简便快速,能够在等温条件下进行,特异性好,具有更高的灵敏性。结论RT-LAMP方法具有灵敏度高,特异性强,操作简便快速,不需要复杂仪器设备等优点,为临床检测诺如病毒快速简便的新方法。     

微流控芯片及其在病原体检测中的应用

微流控芯片又称芯片实验室,是一种以在微米尺度空间对流体进行操控为主要特征的技术平台。微型化、集成化的微流控芯片具有高效、快速、样品和试剂用量少、节约药品等优点,促进了其在病原体检测中的应用。本文对微流控芯片在病原体检测中的优势及其应用研究进展进行综述。     

刚地弓形虫529 bp重复序列环介导等温扩增检测方法的建立

目的建立高效检测刚地弓形虫(Toxoplama gondii)的环介导等温扩增(LAMP)方法,为弓形虫病诊断提供新的技术手段。方法采用在线引物设计软件Primer Explorer 4.0设计弓形虫529 bp基因LAMP扩增引物,用已构建的pMD-19T-529 bp重组质粒为模板,建立LAMP扩增反应,通过添加环引物、设置不同浓度的甜菜碱和Mg SO_4以及不同反应温度对扩增反应体系和反应条件进行优化。以猪、微小隐孢子虫(Cryptosporidium parvum)、猪等孢球虫(Isospora suis)的基因组DNA和超纯水为对照,pMD-19T-529 bp重组质粒为模板,分别进行优化后的LAMP和PCR扩增,评价LAMP方法的特异性。将pMD-19T-529 bp重组质粒梯度稀释为109~100copies/ml,分别进行优化后的LAMP和PCR扩增,评价LAMP方法的灵敏性。结果设计的LAMP扩增引物可扩增出弓形虫529 bp序列保守区域的目的片段。优化结果显示,最佳反应总体系为25μl,其中,甜菜碱最佳浓度为0.4 mol/L,Mg SO_4最佳浓度为6 mmol/L;添加环引物反应30 min的扩增效率与未添加环引物反应60 min的扩增效率相当,可使反应时间缩短30 min;最佳反应条件为63℃30 min,80℃3 min。特异性检测结果显示,所建立的LAMP方法可特异扩增弓形虫529 bp基因片段,对照均未扩增出目的片段。灵敏性检测结果显示,LAMP方法的最低检出值达10~3 copies/ml,效率为PCR扩增(10~4 copies/ml)的10倍。结论建立了弓形虫529 bp重复序列的LAMP检测方法,该法具有较好的特异性和灵敏性。     

Three layer functional model and energy exchange concept of aging process

Relying on a certain degree of abstraction, we can propose that no particular distinction exists between animate or living matter and inanimate matter. While focusing attention on some specifics, the dividing line between the two can be drawn. The most apparent distinction is in the level of structural and functional organization with the dissimilar streams of ‘energy flow’ between the observed entity and the surrounding environment. In essence, living matter is created from inanimate matter which is organized to contain internal intense energy processes and maintain lower intensity energy exchange processes with the environment. Taking internal and external energy processes into account, we contend in this paper that living matter can be referred to as matter of dissipative structure, with this structure assumed to be a common quality of all living creatures and living matter in general. Interruption of internal energy conversion processes and terminating the controlled energy exchange with the environment leads to degeneration of dissipative structure and reduction of the same to inanimate matter, (gas, liquid and/or solid inanimate substances), and ultimately what can be called ‘death.’ This concept of what we call dissipative nature can be extended from living organisms to social groups of animals, to mankind. An analogy based on the organization of matter provides a basis for a functional model of living entities. The models relies on the parallels among the three central structures of any cell (nucleus, cytoplasm and outer membrane) and the human body (central organs, body fluids along with the connective tissues, and external skin integument). This three-part structural organization may be observed almost universally in nature. It can be observed from the atomic structure to the planetary and intergalactic organizations. This similarity is corroborated by the membrane theory applied to living organisms. According to the energy nature of living matter and the proposed functional model, the decreased integrity of a human body's external envelope membrane is a first cause of the structural degradation and aging of the entire organism. The aging process than progresses externally to internally, as in single cell organisms, suggesting that much of the efforts towards the restoration and maintenance of the mechanisms responsible for structural development should be focused accordingly, on the membrane, i.e., the skin. Numerous reports indicate that all parts of the human body, like: bones, blood with blood vessels, muscles, skin, and so on, have some ability for restoration. Therefore, actual revival of not only aging tissue of the human body's membrane, but the entire human body enclosed within, with all internal organs, might be expected. We assess several aging theories within the context of our model and provide suggestions on how to activate the body's own anti-aging mechanisms and increase longevity. This paper presents some analogies and some distinctions that exist between the living dissipative structure matter and inanimate matter, discusses the aging process and proposes certain aging reversal solutions.     

Unusual responses of nocturnal pineal melatonin synthesis and secretion to swimming: Attempts to define mechanisms

Abstract: The effect of swimming at night on rat pineal melatonin synthesis was compared with that of light exposure at night. Rats were forced to swim at 0030 hr (lights out at 2000 hr) and sacrificed by decapitation 15 and 30 min later, immediately after swimming. Other groups of animals were exposed to white light (650μW/cm²) for 15 and 30 min at same time. Swimming caused a rapid and highly significant drop in the melatonin content in the pineal gland; however, the activity of N-acetyltransferase (NAT), the supposed rate limiting enzyme in the melatonin production, was not changed. Despite the drop in pineal melatonin levels, serum concentrations of the indole remained elevated in the rats that swam. In contrast, melatonin levels in the pineal and serum of light exposed rats fell precipitously, accompanied by a significant suppression of NAT activity. Since we anticipated that the strenuous exercise associated with swimming may induce release of artrial natriuretic peptide (ANP) from the heart, which in turn could cause the release of pineal melatonin, in a second study we injected physiological saline intravenously to stretch the cardiac muscle and release ANP. Three milliliters of normal saline was injected during the day into the jugular vein of anesthetized rats that were pretreated with isoproterenol to stimulate pineal melatonin production. Animals were killed 15 min after the saline injection, and pineal NAT activity and pineal melatonin levels were measured. The saline injections caused no alteration in the elevated levels of either NAT or melatonin. These data suggest that the disparity in pineal NAT activity (which was high) and pineal melatonin (which was low), in animals swum at night, may not be caused by ANP which is released during strenuous exercise such as swimming.     

Melatonin and photoperiodic time measurement: Seasonal breeding in the sheep

Abstract: Well-established circadian physiology supports the view that photoperiodic time measurement utilizes the coincidence between the presence of light and a photosensitive phase of a 'biological clock' to alter reproductive status—the so-called external coincidence model of seasonal breeding. In this review, we examine the mechanism whereby photoperiod interacts with presumed suprachiasmatic nuclei activity to allow endogenous melatonin to normally synchronize reproductive activity to the optimal time of year. The Romney Marsh sheep is particularly explored as an experimental model. It is suggested that the on/off activity of seasonal reproduction may be a robust mechanism able to be predictably manipulated by the judicious use of the light/dark cycle and exogenous melatonin, but firmly based on circadian principles.     

Serological survey of HIV and syphilis in pregnant women in Madagascar

Objectives Peripartal transmission of human immunodeficiency virus (HIV) and Treponema pallidum, the causative agent of syphilis, leads to severe consequences for newborns. Preventive measures require awareness of the maternal infection. Although HIV and syphilis testing in Madagascar could be theoretically carried out within the framework of the national pregnancy follow‐up scheme, the required test kits are rarely available at peripheral health centres. In this study, we screened blood samples of pregnant Madagascan women for HIV and syphilis seroprevalence to estimate the demand for systemic screening in pregnancy. Methods Retrospective anonymous serological analysis for HIV and syphilis was performed in plasma samples from 1232 pregnant women that were taken between May and July 2010 in Ambositra, Ifanadiana, Manakara, Mananjary, Moramanga and Tsiroanomandidy (Madagascar) during pregnancy follow‐up. Screening was based on Treponema pallidum haemagglutination tests for syphilis and rapid tests for HIV, with confirmation of positive screening results on line assays. Results Out of 1232 pregnant women, none were seropositive for HIV and 37 (3%) were seropositive for Treponema pallidum. Conclusions Our findings are in line with previous studies that describe considerable syphilis prevalence in the rural Madagascan population. The results suggest a need for screening to prevent peripartal Treponema pallidum transmission, while HIV is still rare. If they are known, Treponema pallidum infections can be easily, safely and inexpensively treated even in pregnancy to reduce the risk of transmission.     

Variabilité des concentrations plasmatiques de l’angiotensinogène et risque d’hypertension artérielle chez le rat transgénique

Aim

Genetic polymorphisms of the human angiotensinogen gene are frequent and may induce up to 30% increase of plasma angiotensinogen concentrations with a blood pressure increase of up to 5 mmHg. Their role for the pathogenesis of human arterial hypertension remains unclear. High plasma angiotensinogen levels could increase the sensitivity to other blood pressure stressors.

Methods

Male transgenic rats with a 9-fold increase of plasma angiotensinogen concentrations and male non-transgenic rats aged 10 weeks were treated or not with NG-Nitro-L-arginine-methyl ester for 3 weeks in their drinking water (n = 3/group). Systolic blood pressure and body weight were measured at baseline and at the end of the study when left ventricular weight and ventricular expression of angiotensin I-converting enzyme and procollagen Iα1 were determined (polymerase chain reaction).

Results

At baseline, transgenic rats had +18 mmHg higher bood pressure and –8% lower body weight compared to non-transgenic rats (P < 0.05) without significant changes for the vehicle groups throughout the study (P > 0.05). NG-Nitro-L-arginine-methyl ester increased blood pressure, left ventricular weight and left ventricular weight indexed for body weight by +41%, +17.6% and +18.6% (P < 0.05) in transgenic and +25%, +5.3% and +6.7% (P > 0.05) in non-transgenic rats compared to untreated animals, respectively. Cardiac gene expression showed no differences between groups (P > 0.05).

Conclusion

Increased plasma angiotensinogen levels may sensitize to additional blood pressure stressors. Our preliminary results point towards an independent role of angiotensinogen in the pathogenesis of human hypertension and associated end-organ damage.     

Morphological and immunocytochemical heterogeneity of cultured pinealocytes from one-week-and two-month-old rats: Planimetric and densitometric investigations

Abstract: In vitro preparations of rat pinealocytes are widely used for biochemical analyses of signal transduction processes. This paper deals with morphological and immunocytochemical features of such preparations. Special attention was paid to the problems of whether pinealocytes represent a heterogeneous cell population and how such heterogeneity may develop during ontogeny. The investigations were performed with cells which were obtained from the pineal organ of one-week-and two-month-old rats, attached to synthetic peptide-coated coverslips or tissue culture chamber slides, and maintained under in vitro conditions overnight. The attached cells were then fixed with paraformaldehyde. These preparations yielded monolayers of spherical cells of different sizes; most cells were isolated, but some of them were aggregated and formed small clusters. On the average, the cells from the one-week-old animals were smaller than the cells from the two-month-old animals. Immunocytochemical demonstration of S-antigen, a pinealocyte-specific marker, showed that the majority of the cells from two-month-old animals were intensely or moderately labelled. Pinealocytes from one-week-old animals were less S-antigen immunoreactive. Only very few cells (less than 1% displayed glial fibrillary acidic protein (GFAP)-immunoreactivity. Planimetric investigations of the cell size and semiquantitative densitometric investigations of the intensity of the S-antigen immunoreaction revealed that (i) pinealocytes kept in vitro form a heterogeneous cell population, and that (ii) this heterogeneity increases during postnatal development from one-week-old to two-month-old animals. Two groups of pinealocytes can be distinguished based on their developmental fate: pinealocytes of one group grow dramatically, but show only a moderate increase in S-antigen immunoreactivity, and pinealocytes of the other group retain their size, but display a distinct increment in S-antigen immunoreacti vitv.     

Effects of pinealectomy and melatonin administration on certain indices of ovarian hyperplasia and/or hypertrophy in rats with both ovaries intact or after unilateral ovariectomy

Abstract: In earlier studies from other laboratories it was shown that melatonin decreased ovarian weight in rats and inhibited compensatory hypertrophy of the remaining ovary after unilateral ovariectomy. This study was designed to examine the influence of melatonin on certain indices of ovarian hyperplasia and/or hypertrophy in adult female rats with both ovaries preserved and with either an intact pineal gland or with the pineal gland removed (pinealectomy, PX) or, finally, in sham-PX animals. Similar studies were conducted on rats after unilateral ovariectomy, referring the examined parameters to the remaining intact ovary. The studies included mitotic activity of granulosa layer cells and corpus luteum cells, ovarian weight, ovarian cross-sectional area, cross-sectional area of the granulosa layer of all the Graafian follicles and the cross-sectional areas of the corpora lutea, visible on the ovarian cross-section. On the basis of results, we conclude that: 1) the effect of PX on the processes of ovarian hyperplasia and hypertrophy may vary; analogously, exogenous melatonin administration may influence ovarian hyperplasia and hypertrophy in different ways; 2) PX and exogenous melatonin may, under certain conditions, exert similar biological effects, even synergistic effects; 3) melatonin inhibits ovarian growth processes, while the effects of PX are variable; 4) the results indicate that in experiments performed on rats, with the use of two control groups, i.e., intact and sham-PX, melatonin effects on these two groups may differ.