细菌通用引物PCR快速诊断全身性感染的研究

目的:建立PCR直接检测临床血标本中病原菌的方法,探索其快速诊断全身性感染(sepsis)的应用价值.方法:利用16S rRNA基因的高度保守性,设计并合成细菌的通用引物;采用合成的引物扩增标准菌株及全身性感染病人的血样本,并与血培养进行对照.结果:通用引物PCR可检出0.005pg的大肠埃希菌标准菌株DNA;临床分离的大肠埃希菌、金黄色葡萄球菌PCR扩增产物于255bp处均可见清晰电泳条带,正常人无菌血白细胞及白色念珠菌DNA扩增产物均未见电泳条带出现;PCR可快速地检出血液中多种病原菌DNA,所检的8例病人中4例阳性(50%).结论:165 rRNA基因为基础的PCR,可直接用于临床血标本病原菌的检测,初步实验显示其具有特异、快速等优点,敏感性较血培养有增高趋势,有利于重症急性胰腺炎及其他外科危重病Sepsis的早期诊断.     

胆囊结石患者胆汁需氧菌和厌氧菌脱氧核糖核酸检测及意义

目的:为探索提高胆囊结石患者胆汁细菌检出阳性率,从分子生物学水平研究胆囊结石胆汁细菌脱氧核糖核酸(DNA)。方法:采用聚合酶链式反应(PCR)对胆汁中5种需氧菌和4种厌氧菌DNA进行检测。结果:检测70例其中需氧菌DNA存在56例(80％),厌氧菌DNA存在23例(32.9％)。两类细菌DNA共同存在为20例(28.6％)。从21例胆汁中检测出幽门螺杆菌DNA。结论:胆结石刖汁中存在多种细菌DNA。PCR可提高细菌检出阳性率。     

血液中细菌DNA检测——一种外科病人菌血症或细菌易位的敏感诊断方法

本研究对应用聚合酶联反应法(PCR)作血中细菌DNA检测来诊断外科手术病人全身感染和(或)肠道细菌易位的敏感性进行了观察。血标本分别取自30例健康志愿者和30例外科ICU病人。30例健康志愿者中,10例为健康捐肾者,20例为健康成人;30例外科ICU病人中,8例为OKT3组(即器官移植后接受抗CD3单克隆抗体治疗),22例为外科组(包括脓毒血症、感染和无感染)。取全血200～400μl提取DNA,选用以下3对合成的寡核苷酸作引物进行PCR扩增:①大肠杆菌β-半乳糖苷酶基因产生的BG-1和BG-4;②在12种不同的细菌中均有发现的16S rRNA+和16S rRNA-;③发现于     

抗生素治疗大鼠烧伤绿脓杆菌菌血症时释放内毒素的实验研究

目的 观察不同种类抗生素治疗烧伤绿脓杆菌(PA103)菌血症时诱导细菌释放内毒素的情况。方法利用内毒素微量检测技术,检测抗生素治疗烧伤菌血症大鼠血中内毒素水平及血中细菌菌量。结果 应用敏感抗生素治疗烧伤菌血症均能有效杀灭细菌,但同时能不同程度诱导细菌释放内毒素,伊米配能诱导释放内毒素较少,头孢哌酮次之,头孢他啶和头孢氨噻肟较多。结论 不同种类抗生素具有不同程度的诱导绿脓杆菌释放内毒素的作用,释放的量与其杀菌能力无相关。     

实时PCR法在慢性前列腺炎病原学诊断中的应用研究

目的为研究前列腺细菌感染和慢性前列腺炎之间潜在的关联,本文报告了应用实时PCR方法检测前列腺液(EPS)或按摩前列腺后的尿液(VB3)中的细菌。方法细菌模板DNA取自慢性前列腺炎患者的EPS或VB_3标本。1对通用引物:用于扩增所有已知细菌的核糖体DNA(16SrDNA)。3个荧光探针:一个为检测所有细菌的通用探针(Uniprobe);1个为检测大肠杆菌的特异性探针(Ecprobe);1个为检测金黄色葡萄球菌的特异性探针(SAprobe)。结果应用实时PCR方法检测慢性前列腺炎患者,80个标本中有28个检测到细菌,包括14个为大肠杆菌阳性,6个为金黄色葡萄球菌阳性。结论实时PCR具有能定量、快速、特异等优点,有望成为能对慢性前列腺炎患者EPS或VB_3中的细菌进行定性、定量检测的一种很有前途的方法。     

消化道重建术后肠道细菌移位的临床研究

目的：探讨消化道重建术后肠黏膜屏障损伤与肠道细菌移位（BT）及BT与术后全身炎症反应综合征（SIRS）的关系。方法：选择60例择期行消化道重建术的患者,于术前和术后1、3、5 d采集外周血,进行血浆二胺氧化酶及全血细菌DNA检测。全血DNA提取后进行PCR扩增,采用的靶基因为大肠杆菌特异性β半乳糖苷酶基因和16SrRNA基因。观察患者至术后10 d以监测SIRS情况。结果：术前PCR检测全血细菌DNA均为阴性,术后共有14例阳性。23例患者术后发生SIRS,其中12例患者PCR阳性。PCR阳性组SIRS发生率为85.7%（12/14）,阴性组为23.9%（11/46）（P〈0.01）。术后出现SIRS的患者PCR阳性率为52.2%（12/23）,无SIRS组为5.4%（2/37）（P〈0.01）。PCR阳性的患者血浆二胺氧化酶浓度较PCR阴性者明显升高（P〈0.01）,有SIRS的患者血浆二胺氧化酶较无SIRS患者明显升高（P〈0.01）。结论：消化道重建术后BT与肠黏膜屏障损伤密切相关,术后SIRS与BT密切相关。PCR技术可早期诊断细菌移位,对术后SIRS有较好的早期预警价值。     

TaqMan荧光定量PCR检测HIV-1 DNA方法的建立及初步应用

目的建立基于TaqMan探针的HIV DNA荧光定量PCR检测方法,并检验其可靠性和稳定性。方法选择HIV-1的保守区域LTR-gag设计引物和探针,构建质粒标准品,建立检测方法,对12份HIV-1阳性标本和5份HIV-1阴性标本进行检测。将检测结果与罗氏HIV DNA定性检测试剂盒作比较。结果荧光定量PCR体系的检测下限为50拷贝/μl,标准曲线的相关系数为0.99,斜率为-1.65,截距为39.80;12份HIV-1阳性标本,检出率为100%;5份HIV-1阴性标本,检测结果均为阴性。该方法与罗氏HIV DNA定性检测试剂盒检测结果完全一致。采用不同方法提取的DNA再行HIV DNA检测,其差异无统计学意义(t=0.033、P=0.974)。结论成功建立了一种HIV DNA的检测方法,具有快速、特异性强以及稳定性好等优点。     

多聚酶链反应检测细菌DNA对大鼠空、回肠吻合口瘘的早期诊断价值

目的:探讨PCR检测大鼠外周血及腹水中细菌DNA对空肠-空肠、回肠-回肠吻合口瘘的早期诊断价值。方法:健康Wistar雌性大鼠50只，随机分成5组，每组10只：A组为假手术组;B组为空肠-空肠吻合组;C组为空肠吻合口瘘组;D组为回肠-回肠吻合组;E组为回肠吻合口瘘组。采集手术前后外周血及术后腹水，抽提DNA, 比较lacZ基因和16SrRNA基因的PCR阳性率，并观察各组的病理学情况。结果:（1）C，E组术后外周血lacZ基因PCR阳性率与B，D组无显著性差异（P>0.05）;C，E组术后外周血16SrRNA基因PCR阳性率显著高于B，D组（P<0.05）。（2）C，E组腹水lacZ基因和16SrRNA基因PCR阳性率均显著高于B，D组（P<0.05）。（3）C，E组腹水lacZ基因阳性率显著高于外周血（P<0.05）;C，E组腹水16SrRNA基因阳性率与外周血无显著性差异（P>0.05）。结论:（1）PCR检测术后外周血16SrRNA基因对空、回肠吻合口瘘的早期诊断有一定意义;（2）检测术后腹水lacZ基因和16SrRNA基因对空肠-空肠、回肠-回肠吻合口瘘的早期诊断也有一定意义。     

应用巢式PCR扩增孕妇血浆中胎儿SRY基因的研究

目的:建立一种巢式聚合酶链反应(PCR),利用孕妇血浆中的游离胎儿DNA(fetal DNA)鉴定胎儿SRY基因。方法:随机采集30例孕妇外周血标本,采用酚/氯仿法从孕妇血浆中提取DNA,设计两对引物对SRY基因进行巢式PCR扩增,扩增产物经测序加以确认。结果:17例孕男胎的孕妇血浆中有15例经巢式PCR扩增检出SRY基因,而13例孕女胎的孕妇血浆没有检出阳性结果。准确性和敏感性分别为93.3%(28/30)和88.2%(15/17)。结论:应用酚/氯仿法从孕妇血浆中提取游离DNA简单有效,结合巢式PCR扩增SRY基因技术可用于无创性产前性连锁遗传性疾病的诊断。     

反向聚合酶链反应检测肝细胞癌中乙型肝炎病毒DNA整合

目的应用反向聚合酶链反应(inverse polymerase chain reaction,IPCR)法检测肝细胞癌(HCC)中乙型肝炎病毒(HBV)DNA整合.方法提取手术切除石蜡包埋人肝癌组织中DNA,根据IPCR原理,选用HBV DR1区无酶切位点的限制性内切酶酶切,通过连接反应形成环化DNA分子,以此作为模板进行PCR扩增得到已知序列的旁侧序列.琼脂糖凝胶电泳观察PCR扩增产物片段.结果 19例HCC标本中有14例检出整合型HBV DNA,2例同时存在游离型HBV DNA.结论 IPCR可以准确测定肝细胞中HBV DNA的整合.该方法为研究HBV DNA在肝细胞中的整合机制提供一简单、快速、经济途径.     

The detection of microbial DNA in the blood: a sensitive method for diagnosing bacteremia and/or bacterial translocation in surgical patients.

OBJECTIVE: The purpose was to determine the sensitivity of detecting microbial DNA in the blood of surgical patients as a measure for diagnosing systemic infection and/or translocation from the gut. SUMMARY BACKGROUND DATA: Microbial infections and translocation of intestinal bacteria are thought to contribute to multiple system organ failure, but bacterial cultures are often negative in patients with this complication. METHODS: DNA was extracted from the blood of 40 surgical patients and 20 healthy controls. Polymerase chain reaction (PCR) techniques were used to amplify genes from Escherichia coli, Bacteroides fragilis, and a region of 16S ribosomal RNA found in many gram-positive and -negative bacteria. RESULTS: Bacterial DNA genes were not detected in healthy volunteers but were found in all patients with positive blood cultures. All eight transplant patients receiving OKT3 therapy had microbial DNA in their blood, possibly indicating translocation from the gut. Sixty-four percent of critically ill patients had microbial DNA detected in their blood, but only 3 (14%) had positive blood cultures. CONCLUSIONS: The PCR method is more sensitive than blood cultures for detecting bacterial components in the blood of critically ill surgical patients and may detect microbial translocation from the intestine.     

Comparison of polymerase chain reaction and bacteria culture in peritoneal dialysis associated peritonitis

Objective To evaluate clinical application value of polymerase chain reaction (PCR) detection for bacteria in peritoneal dialysis associated peritonitis (PDAP). Methods Peritoneal dialysis fluid specimens were collected from January 2014 to December 2014 in The First Affiliated Hospital of Anhui Medical University. Conventional bacterial culture and PCR detection were used respectively. According to the bacterial 16S rRNA gene, universal primers were devised and designed, based on reference, the specific primers of 17 kinds of experimental bacteria. Real-time fluorescent PCR (Real-time PCR, qPCR) amplification was implemented. The establishment of standard strain DNA extract was used as positive control; sterile double distilled water was used as negative control. Results (1) The traditional bacterial culture results showed that positive proportion was 26/40 in specimen of 40 cases, gram-positive strains accounting for 18/26. Main species were epidermis staphylococcus (5/26), hemolysis staphylococcus (4/26), escherichia coli (4/26), and streptococcus viridans (3/26). (2) The PCR detection results showed that total positive rate was 33/40 in 40 patients specimens, among which 2 cases of positive samples ended up with no specific strains being detected; the main bacteria strains in PCR were not different from ordinary culture results. (3) With bacterial culture as the gold standard, the detection sensitivity of PCR technology for PDAP pathogenic bacteria was 96.15% and specificity was 42.86%; the detection positive rate was significantly higher than ordinary culture method. (4) PCR technology for detecting pathogenic bacteria could produce results within 4-6 hours, while reported positive results in the traditional bacterial culture would take (77.88±15.53) hours, which was significantly longer than PCR. Conclusion Compared with traditional bacteria culture method, PCR method is more sensitive, simple, and quick. Bacteria detection using PCR technique is of clinic applied value in PDRP.     

Polymerase chain reaction for the detection of bacteremia.

Analysis of blood by polymerase chain reaction (PCR) is a more rapid and sensitive method to detect bacteremia than blood culture. The PCR was performed on blood obtained from patients during blood culture draws and on blood from normal volunteers. Eighty-seven patients provided 125 blood samples for blood culture comparison with PCR. Specific PCR primers for Staphylococcus aureus and Escherichia coli that targeted conserved regions common to gram-positive and gram-negative bacteria were used. Selective stringency conditions identified other gram-positive and gram-negative bacteria. The blood culture agreed with the PCR in 93 of the 125 patient specimens (74%). In 29 of these specimens the PCR was positive yet the blood culture was negative. When clinical information was included with positive blood culture to define sepsis in these patients and their specimens were added to the positive blood cultures the statistical accuracy of PCR was 93 per cent. Only three of the 78 specimens with negative PCR had positive blood cultures. The PCR was negative in all but one of the 50 volunteers. PCR is more sensitive than blood culture, and it can quickly rule out bacteremia.     

Detection and identification of oxalate-degrading bacteria in human feces.

BACKGROUND: Oxalate is detoxified (catabolized) via the action of two enzymatic proteins, formyl coenzyme A transferase (encoded by the frc gene) and oxalyl coenzyme A decarboxylase (encoded by the oxc gene), contained in the cytosol of Oxalobacter formigenes that colonizes the human intestinal tract. It is speculated that oxalate-degrading bacteria decrease oxalate absorption from the intestines and their absence in the gastrointestinal tract correlates with the formation of calcium-oxalate urolithiasis. METHODS: Two methods of detection and identification of this bacterial strain were studied in human fecal samples collected from Japanese subjects. Genomic DNA was isolated from bacterial culture, and specific 16S rDNA was amplified by polymerase chain reaction (PCR) followed by sequencing. The oxc gene was amplified directly from human feces by PCR using the specific primers. RESULTS: Oxalate-degrading bacteria were identified by comparing the sequences of 16S rDNA. The oxc gene was directly detected from human feces by PCR. It was ascertained that a combined PCR detection method using both 16S rDNA and the oxc gene allows for identification of O. formigenes in human fecal samples. CONCLUSION: This detection and identification method of oxalate-degrading bacteria using 16S rDNA and oxc gene should be applied in examination of clinical samples.     

Detection of Microbial DNA in the Blood of Surgical Patients for Diagnosing Bacterial Translocation

Bacterial translocation sometimes occurs in patients during surgical stress and is associated with an increased incidence of septic morbidity. However, no reliable method has been established for diagnosing bacterial translocation in humans. Identification of minute quantities of microbial-specific DNA has been made possible using polymerase chain reaction (PCR) techniques. The aims of this study were to determine the prevalence of bacterial translocation in patients with surgical stress using PCR techniques and to evaluate the usefulness of blood PCR techniques for diagnosing bacterial translocation. DNA was extracted from the blood of 52 surgical patients (24 elective major surgery patients and 28 septic patients) and 10 healthy controls. PCR techniques were used to amplify genes from Escherichia coli, Bacteroides fragilis, a region of 16S ribosomal RNA found in many gram-positive and gram-negative bacteria, and Candida albicans. Bacterial and Candida albicans DNA were not detected in healthy volunteers. Enteric bacterial DNA was detected in patients with hepatic lobectomy, and Candida albicans DNA was detected in patients with esophagectomy on the first postoperative day. Enteric bacterial and Candida albicans DNA were detected in septic patients with findings diagnostic of bacterial translocation, such as small bowel obstruction, ulcerative colitis, or supramesenteric arterial occlusion or in those who had undergone chemotherapy for advanced colon cancer. However, none of the patients were positive by the blood culture technique. The PCR method is more sensitive than blood cultures for detecting bacterial components in the blood of septic patients and is a valuable tool for verifying bacterial translocation in patients who have undergone hepatic lobectomy or esophagectomy. It is also valuable in septic patients who do not have a defined focus of infection.     

慢性非细菌性前列腺炎患者前列腺液16SrRNA基因的检测及其临床意义

目的 探讨应用聚合酶链反应（PCR）检测慢性非细菌性前列腺炎（CPPS）患者前列腺液（EPS）中细菌基因,并与CPPS患者的临床治疗效果做相关性分析。方法 以16SrRNA基因为靶序列,设计引物及寡核苷酸探针,采用PCR法检测标准菌株及135例CPPS患者前列腺液中细菌基因。所有CPPS患者均应用抗生素和中成药规范治疗,每2周为1疗程,每2周复查EPS常规1次,必要时适当调整药物。治疗3个月时,进行疗效评估。结果 135例CPPS患者中16SrRNA检测结果阳性为78例,阳性率为57．78％。16SrRNA阳性组患者有效率84．6％,阴性组有效率52．6％;16SrRNA阳性组患者疗效明显优于阴性组。结论 大部分CPPS患者的前列腺液中可以检测到细菌的16SrRNA基因,提示细菌感染在CPPS的发病中有重要作用。     

Inflammatory consequences of the translocation of bacteria and endotoxin to mesenteric lymph nodes

BACKGROUND: Translocation of intestinal bacteria to mesenteric lymph nodes (MLNs) has been documented in humans under a variety of circumstances, yet its clinical significance remains to be established. The aim of this study was to correlate detectable translocation to MLNs of bacteria and endotoxin with local and systemic signs of inflammation. METHODS: From each of 10 patients with carcinoma of the cecal region two MLNs were harvested prior to resection. The presence of bacteria and endotoxin in the lymphatic tissue and blood was determined by culture methods and DNA preparation (PCR) and by a Limulus assay, respectively. Inflammatory mediators were determined in plasma and in MLN homogenates. RESULTS: Viable bacteria were detected in MLNs of 7 patients and in 9 of 20 lymph nodes. PCR revealed traces of bacteria in 4 patients and in 6 of their MLNs. Combining both modalities, the translocation rate was 80% and 55% for patients and MLNs, respectively. There was no detectable bacteremia. Endotoxin was found in the plasma of 7 patients and in 9 MLNs from 5 patients. There was no correlation between culture findings and endotoxin concentrations. Moreover, bacteriological data did not correspond to local or systemic inflammation. The group of MLN with detectable endotoxin differed significantly from LPS-negative nodes with respect to interleukin-6, interleukin-10, and sCD14. Systemic concentrations of endotoxin and inflammatory parameters did not correspond to levels within MLNs. CONCLUSION: Translocation to MLNs occurs in patients with cecal carcinoma. This, however, seems not to be of major clinical significance if no additional physiologic insults are encountered. Irrespective of the presence of bacteria, there are variations in inflammatory reactions between lymph nodes from one and the same patient, probably reflecting fluctuating response mechanisms to low-grade translocation.     

胆固醇结石患者胆囊细菌感染的研究

目的 比较胆固醇类结石患者和非胆石症患者胆道细菌感染情况.方法 用不依赖细菌培养的半定量PCR和16SrRNA序列对照法,检测76例胆固醇类结石患者胆囊黏膜、胆汁和胆石中细菌DNA,与34例非胆石患者对照.结果 胆石组和非胆石组胆汁细菌DNA阳性率分别为77%和67%,胆囊黏膜细菌DNA阳性率分别为64%和69%,两组间差异均无统计学意义(P＞0.05).胆石组细菌种类主要为大肠杆菌、铜绿假单胞菌、金黄色葡萄球菌、不动杆菌、链球菌、鞘氨醇单胞菌、脆弱类杆菌和痤疮丙酸杆菌,胆石组菌种比非胆石组丰富,非胆石组菌种基本上均能在胆石组中找到.结论 胆固醇结石与非胆石症患者胆道细菌感染率相似,胆固醇结石中存在细菌不足以证明细菌参与胆固醇结石的形成.