基因芯片检测肠道致病菌技术的建立和应用

目的 快速、准确地检测水中存在的肠道致病菌。方法 采用基因芯片技术对水中常见肠道致病菌进行检测和鉴定，包括检测靶基冈的扩增、基因芯片的制备、杂交反应和杂交结果的检测与分析四个步骤。结果 应用基因芯片对涉及9个菌属的143株致病菌在相同的条件下进行了杂交检测，得到菌属(科)特异性的杂交图谱，从而达到对细菌进行检测鉴定的目的。结论 制备的基因芯片能够同时检测沙门菌属、志贺菌属、埃希菌属、葡萄球菌属、变形杆菌属的细菌以及小肠结肠炎耶尔森菌、副溶血性弧菌等。     

基因芯片检测7种常见腹泻病致病菌方法研究

目的 建立运用多重PCR和基因芯片技术一次检测腹泻病粪便同一标本中7种常见致病菌的方法。方法 筛选7种常见腹泻病致病菌的特异基因作为检测目的基因,并据此设计引物及探针,制备相应的寡核苷酸芯片,检测了标准菌株, 并直接检测腹泻病粪便标本64份。结果 以多重PCR扩增出7种腹泻病致病菌特异的致病基因片段,并与基因芯片相应的探针杂交。最低检测菌数为10到100cfu/ml。64份腹泻病粪便标本检测出45份致病菌。其中33份志贺菌、12份副溶血弧菌。敏感性高于粪便培养方法。结论 该基因芯片直接检测腹泻病致病菌,具有较高的敏感性和特异性,达到了高通量即高效的检测目的,具有较好的实用性。     

基因芯片技术在常见肠道致病菌感染检测中的应用

目的 建立一种运用PCR结合基因芯片技术检测和鉴定引起腹泻的常见肠道致病菌方法.方法 根据生物信息学技术以细菌16S rDNA和23S rDNA序列设计各细菌的特异型探针和引物,在基因芯片制备基础上,通过对靶基因的扩增、杂交和对杂交结果的分析,对常见肠道致病菌的检测和鉴定效果进行评价,同时对临床采集100份腹泻患者粪便标本通过细菌培养结果,比较基因芯片技术的灵敏性和特异性.结果 100份临床粪便标本中,采用基因芯片技术共检出50份(50％)携带肠道致病菌;细菌培养鉴定出47份(47％)携带肠道致病菌,其中42份基因芯片与细菌培养结果一致,准确率为79.25％,与基因芯片比较差异无统计学意义(x2=5.28,P＞0.05).结论 我们所建立的基因芯片系统具有高通量和高准确性,可以作为临床鉴定细菌的一个重要的工具,而且适用于流行病学调查研究.     

寡核苷酸芯片技术检测5种临床肠道病原菌的研究

背景：寡核苷酸芯片是一种检测病原菌的新技术，具有高通量、特异性等特点。目的：采用寡核苷酸芯片技术建立对5种临床常见肠道病原菌（霍乱弧菌、空肠弯曲杆菌、志贺菌、耶尔森菌、单核细胞增生利斯特菌）进行快速、准确鉴定和检测的方法。方法：选取16SrDNA作为检测靶基因，设计通用引物和寡核苷酸探针。病原菌基因组DNA通过16SrDNA通用引物行聚合酶链反应（PCR），扩增后与芯片上的探针杂交。结果：霍乱弧菌、空肠弯曲杆菌、志贺菌、耶尔森菌、单核细胞增生利斯特菌的杂交信号均很强，其他细菌无杂交信号。寡核苷酸芯片的检测时间约5h，其敏感性可达10^3CFU／ml。寡核苷酸芯片对23例临床腹泻样本的检测结果与传统细菌学培养结果完全一致。结论：寡核苷酸芯片的特异性、稳定性、重复性均较好，是检测临床肠道病原菌准确、可靠、实用性强的方法。     

16S rRNA基因芯片诊断新生儿败血症

目的建立16SrRNA基因加基因芯片检测新生儿败血症的诊断技术，以提高临床检测细菌的速度及准确性。方法对125例拟诊为败血症的新生儿血标本及部分脑脊液标本进行细菌16SrRNA基因及基因芯片检测．包括DNA提取、设计引物和探针、聚合酶链反应（PCR）扩增、基因芯片的制备、杂交、激光扫描与读片。结果125例中PCR检测血标本阳性64例，占51．2％；血培养阳性32例，占25．6％；非特异性指标41例，占32．8％，差异有统计学意义（P〈0．01）。若以血培养阳性和／或非特异指标至少两项阳性为诊断标准，PCR灵敏度为90．3％（56／62），特异度为87．3％（55／63），正确诊断指数为0．776。3例脑脊液标本中PCR阳性2例，培养阳性仅1例。对64例PCR阳性血标本进一步作基因芯片检测，结果通用探针均阳性。其中G^＋探针阳性60份。G探针阳性4份。30份PCR和血培养均阳性的标本中其探针菌株与血培养细菌阳性结果相符。2例脑脊液标本PCR阳性，基因芯片检测结果与培养结果相符。结论168 rRNA基因PCR加基因芯片杂交可为新生儿败血症提供早期、敏感的病原学诊断依据。     

用DNA芯片快速检测结核分支杆菌对异烟肼的耐药性

目的 研制一种新型DNA芯片，用于结核分支杆菌对异烟肼耐药性的快速检测。方法 根据结核分支杆菌与异烟肼耐药性有关的三个基因的序列信息设计信息设计探针并制作DNA芯片，PCR扩增并荧光标记包含突变热点的目的基因片段，与芯片杂交，同时与临床背景资料及DNA测序法相对照。结果 随机抽取5株结核分支杆菌敏感株和40株结核分支杆菌耐异烟肼菌株进行检测，其中5株敏感株均未发生突变，4株耐药株中有29株发生突变，覆盖率达到72．5％；另抽取10株进行DNA测序（包括2株未检测到信号的耐药株），与芯片检测结果完全相符。结论 用DNA芯片检测结核分支杆菌对异烟肼的耐药性具有较高的特异性和灵敏度，该法快速，精确，可以应用于结核病临床药性检测。     

基因芯片法对苏州市结核临床分离株RFP耐药性快速检测价值的研究

目的 通过基因芯片法检测结核分离株RFP的耐药性,为在临床诊断中利用该法快速鉴别苏州市结核RFP耐药菌株的可靠性奠定基础.方法 收集痰液标本45例,利用罗氏固体培养法（金标准）对结核分枝杆菌进行传统药敏试验;利用晶芯结核分枝杆菌耐药检测试剂盒及相关仪器通过核酸提取、PCR扩增、芯片杂交和芯片扫描,对结核分枝杆菌RFP耐药相关基因rpoB的rpoB-RRDR 511,513,516,526,531和533位点进行检测,将基因芯片检测获得的RFP药敏判读结果与金标准法获得的结果进行比较分析;将17例经基因芯片检测过的菌株用PCR法扩增包含rpoB-RRDR的基因片段,经纯化后进行DNA直接测序,将基因芯片对rpoB-RRDR 526,531位点的检测结果与DNA测序获得的结果进行比较分析.结果 与金标准法相比,基因芯片法对菌株RFP药敏检测的准确率为93.3%（42/45）;与DNA测序法相比,基因芯片法对rpoB-RRDR 526,531位点突变型检测的准确率为82.4%（14/17）.结论 rpoB-RRDR 526,531位点突变是导致苏州市结核病RFP耐药的主要因素,通过基因芯片法对rpoB基因相关位点的检测可为临床对RFP耐药结核鉴定提供一种快速、准确和批量性的检测手段,具有极大的应用价值.     

分子信标探针技术用于PCR检测诺卡氏菌SecA1基因

目的将诺卡氏菌属细菌的一段特异性DNA设计成分子信标探针,用于该细菌的PCR检测。方法将诺卡氏菌属细菌、戈登氏菌属细菌及红球菌属细菌菌株分别接种于脑心浸液琼脂培养基分离培养,观察其生长情况,提取菌株DNA作为扩增模板;设计诺卡氏菌属细菌基于secA1基因的特异性分子信标探针,在实时荧光定量PCR反应译体系中加入分子信标探针,PCR产物进行荧光信号检测。结果诺卡氏菌secA1基因经实时荧光定量PCR扩增后可产生阳性荧光信号,红球菌属细菌及戈登氏菌属细菌的secA1基因、阴性对照实验组及空白对照组经实时荧光定量PCR扩增后不产生荧光信号,为阴性。结论 secA1作为看家基因,是用来进行种水平的鉴定及系统进化研究非常理想的靶分子,而分子信标探针技术可以准确、快速、灵敏的进行诺卡氏菌secA1基因检测。     

口腔常见厌氧性细菌感染检测基因芯片的研制

目的研制一种用于检测口腔常见无芽胞厌氧性细菌感染的基因芯片。方法选择口腔感染中常见的无芽胞厌氧性细菌作为菌种,将16S rDNA作为靶基因。借助生物信息学方法,设计通用引物和特异性探针,选择与反义引物互补的核酸序列作为阳性质控探针;大肠杆菌16S rDNA基因序列为阴性质控探针,制备微阵列。进行厌氧性细菌DNA的提取、扩增、杂交、结果分析以及微阵列质量检测。结果制备的用于检测口腔常见无芽胞厌氧菌感染的基因芯片特异性、灵敏度、重现性和稳定性均符合实验要求。结论利用生物信息学方法设计的引物和探针合理,制备的芯片可用于口腔常见无芽胞厌氧性细菌感染的诊断。     

细菌感染患者早期病原学分子诊断技术

目的 建立细菌感染患者早期病原及耐药性分子诊断技术，以便尽早实施有效的抗感染治疗方案，改善患者预后。方法 扩增细菌23S rRNA基因序列，同时扩增多种耐药基因，设计探针，通过基因芯片杂交技术识别其差异序列，鉴别细菌，检测耐药基因，用于病原早期诊断和耐药谱测定；收集血培养标本223份，脑脊液、胸水、腹水标本339份，尿液标本514份，比较所建立方法与传统方法的一致性，验证所建立技术的可靠性。结果 基因诊断方法可正确检出常见病原菌，并检测其是否携带mecA、SHV、CTX-M-1组和CTX-M-2组耐药基因。检测血、尿及脑脊液等无菌体液标本时，所建立的快速检测方法与常规方法的符合率分别为96．4％、99．8％和99．7％，总体符合率为99．1％，耐药性检测与传统药物敏感结果的符合率为95．7％，其准确性与常规方法相当，检测时间比常规方法平均减少（2．09±1．15）d。结论 多重PCR-基因芯片杂交技术可用于耐药菌感染的病原早期诊断和耐药感染的同步检测，应用于临床可望改善患者的预后。     

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.     

Changes in connexin43, the gap junction protein of astrocytes, during development of the rat pineal gland

Abstract: The abundance of gap junctions between rat pineal astrocytes formed by connexin43 (Cx43) was studied during development. Levels and distribution of Cx43 were measured by immunoblotting and indirect immunofluorescence, respectively. The amount of Cx43 in cells located within the gland was low until about the 7th postnatal day and increased to adult values between the 14th and 21st days postpartum. Although astrocytes, recognized by their vimentin immunoreactivity, were scarce before birth, they were abundant by the 7th postnatal day suggesting that the low levels of Cx43 found at this age corresponded to a low expression of this protein. Localization of the immunoreactivity to Cx43 and vimentin showed a close correlation, indicating that mature or immature pineal astrocytes form gap junctions made of Cx43. Since Cx43 levels attained their adult values at about the time the innervation and the functional state of the gland reached maturity (2–3 weeks after birth), it is proposed that astrocyte gap junctions are involved in the function of the adult rat pineal gland.     

Conservative management of perforated duodenal diverticulum： A case report and review of the literature

Duodenal diverticula are a relatively common condition. They are asymptomatic, unless they become complicated, with perforation being the rarest but most severe complication. Surgical treatment is the most frequently performed approach. We report the case of a patient with a perforated duodenal diverticulum, which was diagnosed early and treated conservatively with antibiotics and percutaneous drainage of secondary retroperitoneal abscesses. We suggest this method could be an acceptable option for the management of similar cases, provided that the patient is in good general condition and without septic signs.     

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.     

Presence of immunoreactive growth hormone and prolactin in the ovine pineal gland

Abstract: The use of antisera raised against bovine growth hormone (GH) and ovine prolactin (PRL) enabled the detection of related immunoreactive (ir) sequences of proteins in ovine pineal tissue. The isolation of PRL-like ir-material was accomplished using a 0.25 M ammonium sulphate (pH 5.5) extraction followed by ethanol precipitation, whereas the resulting 2.0 M ammonium sulphate (pH 7.0) precipitate contained a GH-like immunoreactivity. Gel chromatography of the GH-like immunoreactivity (Sephadex G-100) indicated the presence of several GH-like fragments ranging in the M_r range of 7,000 to 55,000. Analyses of the PRL-like ir-material found in pineal tissue on HPLC using a TSK 545-DEAE column led to the resolution into a single peak of immunoreactivity. A single peak of activity was also observed following chromatofocusing and hydrophobic interaction chromatography of the ir-peak from the TSK 545-DEAE column. The PRL-like ir-material inhibited the binding of [¹²⁵I]ovine PRL-S14 to anti-ovine PRL antibodies without showing an affinity for binding to anti-rat PRL or anti-bovine GH antibodies. Scatchard analysis of the binding of pineal PRL-like ir-material and pituitary ovine PRL-S14 to liver membranes from day-20 pregnant rats revealed similar affinity constants (K_a of 4.7 ± 0.2 × 10⁹ M^-1). In addition, the replication of Nb 2 Node rat lymphoma cells was stimulated by pineal PRL-like ir-material, an effect known to be specific for lactogenic hormones. The pineal PRL-like immunoreactivity appeared on sodium dodecyl sulfate polyacrylamide gels as a single major band of M_r 24,000. The functional status of PRL-and GH-like ir-material in the ovine pineal remains to be determined, but evidence is presented that the overall protein synthesis rate of the rat pineal responded to circulating concentrations of PRL.     

Microbiology of human immunodeficiency virus anorectal disease

PURPOSE: Individuals who are seropositive for the human immunodeficiency virus are at high risk for opportunistic infection and anorectal disorders. Little prospective information is available regarding anorectal pathogens in these patients. METHODS: One hundred sixty-three HIV-seropositive patients presented to the colorectal clinic between 1989 and 1992. Forty-seven (29 percent) patients were thought to have an infectious process and were prospectively studied using a standardized multiculture protocol. RESULTS: Mean age was 33 (range, 19–59) years. All were male; high-risk behavior accounted for 87 percent of HIV transmissions. Presenting complaints included anorectal pain (79 percent), pus per anum (28 percent), and blood per anum (26 percent). Examination revealed perianal tenderness (60 percent), condyloma (38 percent), perianal ulcers (38 percent), and anal fissures (34 percent). Sixty-six sets of cultures were performed; 28 patients had one set, 15 had two sets, and 4 had three sets. Thirty-two of these 47 patients (68 percent) had positive cultures including herpes (50 percent), cytomegalovirus (25 percent),Neisseria gonorrhoeae (16 percent), chlamydia (16 percent), acidfast bacilli (2 percent), and others (9 percent). Six of 32 patients with positive cultures had more than one organism cultured. Sixteen (50 percent) patients with positive cultures were treated medically, 8 (25 percent) were treated surgically and 8 (25 percent) were treated with both modalities. Sixty-one procedures were performed on 17 patients for condylomata. Eighteen patients had 20 procedures for abscesses, 50 percent of whom had positive cultures for other than common bowel flora; all improved. Fourteen patients underwent 33 procedures for perianal fistulas.Mycobacterium fortuitum was cultured from one patient who required 13 procedures for abscesses and fistulas. Forty-five (96 percent) patients were followed for an average of 12.5 months ±2.9 SEM (range, 1–94 months). Symptoms were improved or resolved in 22 of 32 (69 percent) patients with positive cultures and in 11 of 13 (84 percent) with negative cultures. CONCLUSIONS: Specific pathogens may often be identified in human immunodeficiency virus-seropositive patients with anorectal disorders if aggressively sought. Although patients without specific pathogens identified may be expected to improve with planned empiric treatment, positive identification allows more directed therapy.