应用PCR技术对L型布氏菌的检测

近２０年来，随着分子生物学技术的发展，多聚酶链反应（PCR）技术已广泛应用于各种传染病病原因子的检测犤１犦，而对布氏菌的抗原的检测亦有报道犤２，３犦，但对布氏菌L型DNA的检测未见报道，研究布氏菌L型的生物学特性，对于慢性潜伏型布氏菌病（简称布病）的临床诊断及布病的流行病学都具有很重要的意义。我们应用PCR方法对布氏菌L型提取的DNA进行了检测。实践证明，该方法对于检测布氏菌L型是有效的，图１牛种布氏菌１０４M，１０４M诱变２２代L型及牛种布氏菌非典型株８５００７诱变１０代L型的DNA基因扩增后电泳图１材料…     

牛种布鲁氏菌A19-△VirB12标记疫苗双重实时荧光定量PCR方-法的建立

目的 建立一种区分牛种布鲁氏菌A19-△VirB12标记疫苗株与布鲁氏菌野毒感染株的双重荧光定量PCR方-法。方法 分别以布鲁氏菌4型分泌系统中VirB8基因、VirB12基因序列设计2对引物及探针,优化实时荧光PCR反应体系及条件。以牛种布鲁氏菌A19-△VirB12标记疫苗株、牛种布鲁氏菌A19疫苗株、羊种布鲁氏菌M5疫苗株以及猪种布鲁氏菌S2疫苗株、大肠杆菌、沙门氏菌基因组DNA进行 Realtime-PCR扩增,评价该方-法特异性。分别构建布鲁氏菌VirB12基因和VirB8基因片段阳性质粒,10倍系列稀释后进行Realtime-PCR扩增,测定该方-法的敏感性。结果 本方-法具有良好的特异性,牛种布鲁氏菌A19疫苗株、羊种布鲁氏菌M5疫苗株以及猪种布鲁氏菌S2疫苗株基因组DNA同时出现VirB8基因与VirB12基因阳性扩增,牛种布鲁氏菌A19-△VirB12标记疫苗株仅出现VirB8基因阳性扩增,大肠杆菌、沙门氏菌均未扩增出目的条带,对VirB8基因及VirB12基因片段阳性质粒的检测限分别为约10² copies/μL和10³ copies/μL。该方-法仅用于鉴别区分牛种布鲁氏菌A19-△VirB12标记疫苗株与布鲁氏菌野毒株。结论 本研究建立的布鲁氏菌双重Realtime-PCR方-法,具有良好的特异性和敏感性,为今后鉴别牛种布鲁氏菌A19-△VirB12分子标记疫苗免疫牛与自然感染牛提供技术支撑。     

快速检测牛羊猪种布鲁杆菌多重PCR的建立

目的 建立一种能同时快速检测并能鉴别牛、羊、猪种布鲁杆菌的多重PCR方法.方法 根据IS711插入序列设计1条公共引物和3条牛、羊、猪种布鲁杆菌(544A、16M、1330S)特有序列引物,进行多重PCR反应;选择耶尔森菌O:9、大肠埃希菌O157:H7、鼠伤寒沙门菌47729进行多重PCR反应的特异性检测;倍比稀释定量法观察牛种布鲁杆菌多重PCR反应的敏感性.结果 牛、羊、猪种布鲁杆菌多重PCR反应扩增片段产物长度分别为485、731、248 bp;耶尔森菌O:9、大肠埃希菌O157:H7、鼠伤寒沙门菌47729加入布鲁杆菌中进行多重PCR反应.扩增结果呈阴性;牛种布鲁杆菌多重PCR反应敏感性为0.0967 Pg.结论 成功建立快速检测牛、羊、猪种布鲁杆菌多重PCR扩增反应方法,且其特异性、敏感性较好.     

PCR检测布氏菌DNA的试验研究

目的 探讨编码牛种布氏菌同一蛋白基因序列不同扩增片断（分别为330bp、230bp和223bp）的3对引物检测布氏菌DNA PCR试验的特异性和敏感性。方法 用煮沸法和酚提取法分别从5个种布氏菌和耶尔森氏菌0：3、O：9、大肠菌O：157中提取DNA进行PCR试验。结果 通过此种方法可检测到小到1PG的布氏菌DNA。对布氏菌5个种的典型株进行DNA分析，并获得独有的相应片断。而2种与布氏菌SPP存在血清学交叉反应的革兰氏阴性细菌未检测到特异DNA片段。结论 3对引物均有良好的特异性和敏感性，但P5、P6稳定性稍差。     

流产布氏杆菌的检测及生物型PCR鉴定

目的 确定近年来黑龙江省部分牛场频繁发生奶牛流产的病因. 方法 对发病的4个牛场进行流行病学调查,从流产胎牛真胃内容物中分离布氏杆菌;根据布氏杆菌不同种IS711设计用于鉴别流产布氏杆菌(B. abortus)、猪布氏杆菌(B. suis)和羊布氏杆菌(B. ovis) 的4条引物,应用PCR方法进行生物型鉴定. 结果 从流产胎牛真胃内容物中分离出6株布氏杆菌,经PCR可扩增,产物大小为498 bp.鉴定为流产布氏杆菌. 结论 引起奶牛流产的主要病原为牛流产布氏杆菌.     

布鲁氏菌与耶尔森氏菌感染的鉴别诊断试验研究(Ⅱ)

目的 用PCR和酶联试验鉴别布氏菌与耶尔森氏菌感染。方法 以不同种布氏菌及不同型耶尔森氏菌分别感染动物，不同时间采样进行PCR和DAgS-ELISA及其他血清学方法进行检查。结果 两菌感染血清的SAT都出现了不同程度的交叉，RBPT及DAgS－ELISA几乎无交叉反应。布氏菌与耶尔森氏菌0：9型的B.abortus 36KD蛋白基因引物的PCR有明显交叉反应。结论 用RBPT或DAgS-ELISA对两菌感染的鉴别是有益的。用B.abortus 36KD蛋白基因引物的PCR鉴别两菌感染是困难的，而且有交叉扩增。     

不同PCR方法检测布鲁菌阳性样本的敏感性分析

目的利用布鲁菌分离株及对应的患者血液样本验证不同引物扩增的特异性及其敏感性,为将PCR方法应用于人布鲁菌病临床诊断提供参考。方法对血液样本进行传统布鲁菌分离培养,根据表型对分离株进行种型鉴定;用玻片凝集试验(PAT)和试管凝集试验(SAT)两种血清学方法检测培养阳性的血液模板;应用不同布鲁菌属引物B4/B5、BP26以及羊、牛和猪种引物对布鲁菌分离株进行鉴定,同时对其血液模板进行PCR检测。结果从急性发热患者血液分离到24株布鲁菌,经表型鉴定均为羊种。对分离株进行PCR鉴定,布鲁菌属引物B4/B5、BP26及羊种引物在分离株中均扩增到目的条带。应用不同引物PCR方法对血液模板检测的阳性率依次为B4/B5(79.17%),羊种(66.67%)和BP26(25.00%);B4/B5和羊种引物扩增同为阳性的符合率为41.67%,B4/B5或羊种引物扩增为阳性的符合率为91.67%。结论在布鲁菌分离培养阳性的血液样本中,应用单一引物PCR进行人布鲁菌病诊断的敏感性较低,将布鲁菌属B4/B5和地方流行种引物结合可提高PCR检测方法的敏感性和特异性。     

猪种布鲁氏菌病防治研究进展

布鲁氏菌病(简称布病)是一种人畜共患的传染病,全世界有160个国家和地区存在人畜布病,分布在亚洲、非洲、欧洲、大洋洲和拉丁美洲。布鲁氏菌属包括6个生物种19个生物型。羊种布氏菌(B.militensis)3个生物型,牛种布氏菌(B.abortus)8个生物型,猪种布氏菌(B.suis)5个生物型,犬种布氏菌(B.canis)、绵羊附睾种布氏菌(B.ovis)和沙林鼠种布氏菌(B.neotomae)各1个生物型。我国除沙林鼠种布氏菌外,有5种12个生物型布氏菌存在和流行,以羊种布氏菌流行最严重,疫区面广,其次为牛种布氏菌和猪种布氏菌。80年代由于羊种菌引起布病逐步控制,牛种菌引起布病有相对增多趋势。     

青海省布氏菌菌株的分布和特点

随着布病防治工作的深入,为了解我省布氏菌菌株的分布和特点,对多年来分离的113株布氏菌作了种的鉴定。这些菌株包括有:1954年以来由我省畜牧兽医部门从畜间分离的67株布氏菌;1958年以来由布防工作组在人间分离的41株布氏菌,以及在1978年和1981年由海西州地方病防治所从野生岩羊和黄羊中分离的5株布氏菌。并对其中的59株作了生物型的鉴定。鉴定用的对照菌株有羊种布氏菌16M、牛种布氏菌544A 和猪种布氏菌1330S。鉴定试验选用国内外常用的布氏菌的种和生物型鉴定     

应用分子生物学技术检测布氏菌抗原的研究——Ⅰ 聚合酶链反应检测布氏菌抗原的研究

我们把聚合酶链反应技术应用于布氏菌的检测,探索了该PCR体系扩增的最佳反应条件。它对6种布氏菌纯化DNA均可扩增,同时还测定了PCR的敏感性和特异性。证实该方法最低可检测200个菌细胞。与布氏菌血清学交叉反应较多的耶氏菌等PCR结果阴性,说明这对引物具有很高的特异性。     

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.     

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.     

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.