聚合酶链反应测Vi抗原在伤寒早期诊断中的应用

为选择更为敏感、特异的伤寒早期诊断方法，从伤塞沙门菌表达Ｖｉ抗原的特异结构基因ＶｉａＢ区域选择二对引物，建立了套式－聚合酶链反应（ｎｅｓｔｅｄ－ＰＣＲ）。检测伤寒早期标本３６份、结果血培养阳性２５份（６９％），肥达反应阳性１７份（４７．２％），ＰＣＲ阳性２９份（８０．６％）；２５份血培养阳性标本，ＰＣＲ均为阳性，并从血培养奶性标本中检出Ｖｉ－ＤＮＡ阳性４份。研究表明，应用ｎｅｓｔ－ｅｄ－ＰＣＲ检测     

聚合酶链反应－冷单链构象多态性快速检测结核分支杆菌rpoB基因突变

目的评估ｒｐｏＢ基因突变检测在结核分支杆菌利福平耐药性测定中的应用价值。方法采用聚合酶链反应－冷单链构象多态性（ＰＣＲ－冷ＳＳＣＰ）方法对８７株结核分支杆菌临床分离株及有药敏结果的２２份相应肺结核患者痰标本进行分析。结果ＰＣＲ扩增ｒｐｏＢ基因的敏感性为１００ｐｇＤＮＡ及５０００个菌体，属于分支杆菌特异。所有分离菌的ｒｐｏＢ基因ＰＣＲ扩增均为阳性。采用套式ＰＣＲ可使扩增敏感性提高１００倍。与传统药敏试验方法相比，ＰＣＲ－冷ＳＳＣＰ对８７株结核分支杆菌临床分离菌利福平耐药性的检测敏感性和特异性分别为８９．６％及１００％。２２份涂阳培阳痰标本中套式ＰＣＲ扩增阳性的６份均为高度利福平耐药，其ＳＳＣＰ结果也与相应分离株的药物敏感性试验相符。结论ＰＣＲ－冷ＳＳＣＰ检测结核分支杆菌ｒｐｏＢ基因突变快速、简便、易行，适用于结核分支杆菌分离株利福平耐药性的快速测定。如进一步提高引物增特异性及敏感性，该技术可望用于临床标本直接检测     

套式聚合酶链反应快速诊断结核性心包炎

快速诊断结核性心包炎以便及早抗痨治疗防止缩窄性心包炎的发生。应用套式聚合酶链反应（Ｎｅｓｔｅｄ－ ＰＣＲ） 、直接涂片、培养对８３ 例心包积液标本进行结核菌检测。结果：结核性心包积液Ｎｅｓｔｅｄ － ＰＣＲ阳性率为７２ ．５ ％ 、培养阳性率为１９ ．６ ％ 、涂片镜检阳性率为２３ ．５ ％ 。非结核性心包积液无１ 例阳性。Ｎｅｓｔｅｄ－ ＰＣＲ特异性好、敏感性强并可能避免一般聚合酶链反应（ＰＣＲ） 所出现的假阳性     

聚合酶链反应－单链构象多态性用于耐利福平结核分支杆菌rpoB基因突变的研究

目的研究结核分支杆菌ｒｐｏＢ基因突变及其与利福平（ＲＦＰ）耐药性的关系。方法以参考菌株结核分支杆菌Ｈ３７Ｒｖ为对照，用聚合酶链反应－单链构象多态性（ＰＣＲ－ＳＳＣＰ）方法分析了４０株结核分支杆菌临床分离株。ＰＣＲ－ＳＳＣＰ方法由ＰＣＲ扩增和扩增产物ＤＮＡ单链多态性分析组成。结果两对引物ＰＣＲ扩增产物分别为４１１和２５８ｂｐ，其敏感性分别为５ｐｇ／μｌ、５００个菌／ｍｌ和１ｐｇ／μｌ、５００个菌／ｍｌ；均为属特异性。４０株结核分支杆菌临床分离株２５８ｂｐ扩增片段ＳＳＣＰ图谱的特点：以参考菌株结核分支杆菌Ｈ３７Ｒｖ为对照，１０株敏感株均无区别；单耐ＲＦＰ或包括ＲＦＰ多种抗结核药３０株，除３株外，其余２７株ＳＳＣＰ图谱有明显的区别；检测阳性率为９０％，特异性为１００％。结论ＰＣＲ－ＳＳＣＰ方法可检测出耐ＲＦＰ结核分支杆菌ｒｐｏＢ基因突变；该基因是ＲＦＰ的药物靶编码基因，它的突变与ＲＦＰ耐药性有密切关系，这有助于结核分支杆菌耐药性的快速检测和研究     

结核分支杆菌DNA的单管巢式聚合酶链反应检测

目的探讨单管巢式聚合酶链反应（ＳＮＰＣＲ）检测石蜡包埋组织结核分支杆菌ＤＮＡ的特异性和敏感性。方法应用普通ＰＣＲ（ＧＰＣＲ）、双管巢式ＰＣＲ（ＤＮＰＣＲ）和ＳＮＰＣＲ对结核分支杆菌ＢＣＧ和３０例结核性淋巴结炎石蜡包埋组织进行结核分支杆菌复合群ＩＳ６１１０特异插入序列片段ＤＮＡ检测。结果ＤＮＰＣＲ和ＳＮＰＣＲ检测ＢＣＧＤＮＡ均于１５ｆｇ以上呈现阳性结果，其敏感性明显优于ＧＰＣＲ（４８０ｆｇ）。ＧＰＣＲ、ＤＮＰＣＲ和ＳＮＰＣＲ检测３０例结核性淋巴结炎阳性率分别为４３％、１００％和１００％，抗酸染色阳性率（１０％）与３种ＰＣＲ法相比差异有非常显著意义（均Ｐ＜０．０１）。ＧＰＣＲ阳性率与ＤＮＰＣＲ和ＳＮＰＣＲ相比差异亦具非常显著意义（均Ｐ＜０．０１）。ＳＮＰＣＲ阳性率与ＤＮＰＣＲ相同。结论巢式ＰＣＲ检测淋巴结石蜡包埋组织结核分支杆菌的敏感性显著高于ＧＰＣＲ，其中ＳＮＰＣＲ具有与ＤＮＰＣＲ相同的特异性和敏感性，并具有更大的实用价值。     

耐多药结核病耐药分子机制的研究

目的 研究耐多药结核分支杆菌耐药的分子机理，建立快速检测耐药基因的分子药敏试验方法。方法 通过ＰＣＲ和ＰＣＲ－ＳＳＣＰ分析结构分支杆菌耐多药临床分离株的ｒｐｏＢ、ｒｐｓＬ、ｋａｔＧ基因和ｉｎｈＡ调节序列。结果 ＰＣＲ分析耐药基因的敏感性为１－０ｐｇＤＮＡ；除ｒｐｏＢ基因引物ＰＣＲ扩增为属特异性外，余耐药基因引物ＰＣＲ扩增都具有较高的的特异性。２０株耐多药分离株中，９０％有２种以上耐药遗传标志改变，     

杜氏利什曼原虫SSU rRNA逆转录多聚酶链反应的建立

我们采用引物Ｒ２２２和Ｒ３３３建立杜氏利什曼原虫ＳＳＵｒＲＮＡ逆转录多聚酶链反应（ＲＴ－ＰＣＲ）。采用ＳｉＯ２－高盐吸附法同时抽提杜氏利会曼原虫ＲＮＡ和ＤＮＡ，分别用ＰＣＲ和ＲＴ－ＰＣＲ进行扩增，结果显示以ＲＮＡ为检测对象的ＲＴ－ＰＣＲ肉眼可见区带的检测水平较以ＤＮＡ为检测对象的ＰＣＲ扩增高１００倍，可见ＲＴ－ＰＣＲ具有敏感性高，特异性强的特点。     

杜氏利什曼原虫SSU rRNA逆转录多聚酶链反应的建立

我们采用引物Ｒ２２２和Ｒ３３３建立杜氏利什曼原虫ＳＳＵｒＲＮＡ逆转录多聚酶链反应（ＲＴ－ＰＣＲ）。采用ＳｉＯ２－高盐吸附法同时抽提杜氏利什曼原虫ＲＮＡ和ＤＮＡ，分别用ＰＣＲ和ＲＴ－ＰＣＲ进行扩增，结果显示以ＲＮＡ为检测对象的ＲＴ－ＰＣＲ肉眼可见区带的检测水平较ＬＪＤＮＡ为检测对象的ＰＣＲ扩增高１００倍，可见ＲＴ－ＰＣＲ具有敏感性高、特异性强的特点。     

mip基因聚合酶链反应结合地高辛标记探针杂交鉴定嗜肺军团菌

目的对嗜肺军团菌国外参考菌株及国内分离菌株的巨噬细胞感染增强子（ｍｉｐ）基因分析，建立一种快速、敏感、特异的嗜肺军团菌基因检测鉴定方法。方法利用嗜肺军团菌种特异性ＤＮＡ片段ｍｉｐ基因通过聚合酶链反应（ＰＣＲ）扩增，对ＰＣＲ阳性产物进行地高辛标记的ｍｉｐ基因探针杂交，观察其反应结果。并对２６株环境和患者中分离的疑似嗜肺军团菌菌株进行分析。结果所有实验用嗜肺军团菌国外参考菌株及国内分离菌株的ｍｉｐ基因的ＰＣＲ扩增结果都为阳性，探针杂交结果也为阳性，而非嗜肺军团菌及与非军团菌对照菌株的ＰＣＲ扩增结果及探针杂交结果都为阴性，本实验其灵敏度和特异度均为１００％。另外２６株疑似嗜肺军团菌菌株有６株被鉴定为嗜肺军团菌。结论所建立嗜肺军团菌的快速初步鉴定方法具有很高的特异性和敏感性，并具有较高的应用价值     

PCR—冷SSCP快速检测结核菌rpoB基因突变

为了评估ｒｐｏＢ基因突变检测在结核菌对利福平耐药性测定中的应用价值，作者对８７株结核菌临床分离株及有药敏结果的２２份相应肺结核患者痰标本进行了ＰＣＲ－冷ＳＳＣＰ分析。结果；ＰＣＲ扩增ｒｐｏＢ基因的敏感性１００ｐｇＤＮＡ及５０００个菌体，于分支杆菌特异。     

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.     

The immunoneuroendocrine role of melatonin

Abstract: A tight, physiological link between the pineal gland and the immune system is emerging from a series of experimental studies. This link might reflect the evolutionary connection between self-recognition and reproduction. Pinealectomy or other experimental methods which inhibit melatonin synthesis and secretion induce a state of immunodepression which is counteracted by melatonin. In general, melatonin seems to have an immunoenhancing effect that is particularly apparent in immunodepressive states. The negative effect of acute stress or immunosuppressive pharmacological treatments on various immune parameters are counteracted by melatonin. It seems important to note that one of the main targets of melatonin is the thymus, i.e., the central organ of the immune system. The clinical use of melatonin as an immunotherapeutic agent seems promising in primary and secondary immunodeficiencies as well as in cancer immunotherapy. The immunoenhancing action of melatonin seems to be mediated by T-helper cell-derived opioid peptides as well as by lymphokines and, perhaps, by pituitary hormones. Melatonin-induced-immuno-opioids (MHO) and lymphokines imply the presence of specific binding sites or melatonin receptors on cells of the immune system. On the other hand, lymphokines such as -γ-interferon and interleukin-2 as well as thymic hormones can modulate the synthesis of melatonin in the pineal gland. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ.     

Response of rat pineal melatonin to calcium, magnesium, and lithium is circadian stage dependent

Abstract: Herein we documented the response of pineal melatonin production to electrolytes known to be effective on pineal function in view of a possible circadian stage dependence. We studied the release of melatonin by perifused rat pineal glands at 2 different circadian stages corresponding to the middle of the light and dark periods, i.e., respectively, 7 and 19 HALO (Hours After Light Onset, L:D = 12:12). The initial efflux rates were, as expected, much higher in the perifusates of glands removed from rats sacrificed during the dark phase than of those removed during the light phase. After 3 hr of perifusion, melatonin release reached similar levels which were found constant up to the 8th hr of perifusion, whatever the circadian stage. Perifusion of the glands with physiological concentrations for the rat of calcium (5.2 mmol/1) and magnesium (1.34 mmol/1) resulted in a stimulatory effect on the pineal glands removed from rats sacrificed in the middle of the dark period (19 HALO), whereas no effects were observed on the pineal glands removed from rats sacrificed during the light (7 HALO). Lithium (0.28 and 0.55 mmol/1) was ineffective on melatonin release in pineal glands removed 7 and 19 HALO. Our results show differences in the initial efflux rates of melatonin and in the response of perifused pineal glands to calcium and magnesium according to the circadian stage.     

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.