PCR法检测16S rRNA基因在败血症诊断中的价值

目的评价外周血细菌16S rRNA基因检测诊断败血症的敏感性及特异性。方法利用通用引物对6种常见细菌16S rRNA基因进行扩增，通过阳性菌株及阴性对照检测引物的特异性；观察不同退火温度及不同细菌浓度下PCR检测效果；检测败血症患者及正常人血液标本中的16S rRNA基因表达，并与血培养结果进行比较，评价其诊断意义。结果6种阳性菌株均出现特异阳性条带，阴性质控未出现阳性条带；不同退火温度对PCR结果无明显影响，以55、58℃最佳；PCR方法的最低细菌检测浓度为1．5×10^3cfu／ml；观察组血培养阳性率为38．3％（23／60），血液16S rRNA基因阳性表达率为86．6％（52／60），P〈0．01。结论PCR法检测16S rRNA基因用于败血症诊断特异性及敏感性强，但应注意规范操作、避免污染。     

16SrRNA基因PCR加反相杂交技术检测细菌DNA

目的探讨聚合酶链反应（ＰＣＲ）加反相杂交技术在细菌ＤＮＡ检测中的应用。方法以１６ＳｒＲＮＡ基因为靶序列，设计引物及寡核苷酸探针，采用ＰＣＲ法加反相杂交检测标准菌株及临床标本细菌ＤＮＡ。结果对２０株不同标准菌株进行ＰＣＲ扩增，均出现３７１ｂｐ长度的ＤＮＡ片段，敏感性试验可检测出１０－１２ｇ的细菌ＤＮＡ，与人基因组及病毒无交叉反应；２２份血培养阳性标本及４份脑脊液培养阳性标本均扩增出３７１ｂｐ长度ＤＮＡ条带，反相杂交区分革兰阳性／阴性细菌与培养结果相符。结论１６ＳｒＲＮＡ基因ＰＣＲ加反相杂交技术检测细菌ＤＮＡ，具有敏感、快速、准确的特点，为细菌感染的临床诊断提供了科学的依据。     

一种快速检测患者血液标本猪链球菌方法的研究

目的建立一种快速检测患者血液标本中猪链球菌的方法。方法收集患者血液标本。提取细菌金基因组DNA；根据猪链球菌6个特异基因设计引物，采用PCR技术对这些基因进行检测，并对PCR扩增阳性的基因产物进行序列分析验证，并分析该方法诊断猪链球菌病的特异性和敏感性。结果取唾液链球菌等6种链球菌进行验证，该检测方法的特异性为100％。模拟标本（细菌含量〉10^3个／ml）的敏感性为100％，血培养阳性病人标本的敏感性为100％。通过对90份血培养阴性临床标本的检测，9份PCR结果阳性，序列分析结果证实扩增片段为目的基因。结论该方法灵敏、特异、快速。可直接用于患者血液标本的猪链球菌特异性基因的检测，为猪链球菌感染的早期诊断提供实验室依据。     

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。     

细菌性传染病

可溶性细胞间黏附分子-1、降钙素原在新生儿败血症诊断中的价值，16SrRNA基因PCR加基因芯片杂交快速诊断新生儿败血症，63例医院感染败血症的临床特征分析，静脉免疫球蛋白治疗新生儿败血症45例疗效分析，血前降钙素在新生儿败血症中的应用价值探讨，抗出血性大肠杆菌O157：H7单克隆抗体的制备及鉴定，不同剂量氢化可的松对大肠杆菌致休克早期大鼠肺损伤的影响。     

16SrRNA基因序列分析用于诊断病原性细菌感染初步研究

目的建立一种可适用于多种病原菌检测的方法,以提高病原学诊断率,有利于传染病的病原学监测。方法细菌16SrRNA基因通用引物对直接从血液、尿液、脑脊液标本中提取的细菌DNA进行PCR扩增,并对16SrRNA基因扩增产物进行克隆和序列分析。结果检测了28份临床标本,16SrRNA基因序列分析与尿液细菌培养结果不完全吻合;血液和脑脊液的16SrRNA的检出率均高于细菌培养。结论16SrRNA基因检测分析和细菌培养均具有良好的诊断效果,而针对16SrRNA基因的PCR直接扩增核酸并进行序列分析可对分离培养阴性的标本进行有效的补充诊断,可检测到多种病原菌如肠球菌,大肠杆菌,表皮葡萄球菌,牛链球菌,猪链球菌,布鲁氏菌,表皮葡萄球菌和脑膜炎球菌等,为疾病的诊断提供良好的线索。     

应用16S-23S rRNA基因区间对败血症常见菌的鉴定

目的：建立检测不同菌种细菌的16S－23S rNA基因区间的特异图谱。方法：应用聚合酶链反应（PCR），限制性内切酶片段长度多态性分析（RFLP），分子克隆及测序技术，对临床常见的代表20个属26个种的标准菌株及相应的临床分离菌株共61株进行PCR扩增，同时对临床标本进行培养并与PCR－RFLP比较，探讨其在临床应用中的价值。结果：26株不同的标准菌株行PCR扩增后，分别出现1条带，2条带，3条带及多条带的不同DNA图谱，其敏感性为2．5CFU，与人类基因组DNA，真菌及病毒无交叉反应，其中14种菌经PCR扩增即可区分，另10种经Hinf I或AluI酶切后才能区分，肺炎克雷伯菌与坚韧肠球菌间的差异在第779位碱基上不同，Xma Ⅲ酶能进行区别，临床42例血培养15例阳性，阳性率35．7％；而PCR阳性27例，阳性率达64．3％，其阳性率明显高于血培养（P＜0．01），6例脑脊液标本中1例培养为表皮葡萄球菌，其PCR也阳性，2例培养阴性标本，其PCR也阳性，经图谱分析为葡萄球菌，1例培养为新型隐球菌的脑脊液标本PCR检测为阴性，另2例PCR及培养均阴性。结论：建立了PCR加RFLP技术快速检测细菌16S－23S rRNA基因区间的方法，进一步为临床细菌感染的病原诊断提供了新的科学依据。     

16SrRNA基因检测在老年糖尿病烧伤脓毒血症诊治中的价值

目的探讨16S核糖体RNA(16SrRNA)基因检测对于老年糖尿病患者烧伤脓毒血症耐药性检测的临床价值。方法收集老年糖尿病烧伤患者可疑脓毒血症患者41例外周静脉血标本共45份,将每份标本分别进行血常规、C反应蛋白(CRP)、白细胞介素(IL)-6、-10、肿瘤坏死因子(TNF)-α、空腹血糖(FPG)、糖化血红蛋白(Hb A1c)、血液培养以及16SrRNA基因检测,比较两种检测方法对细菌病原体检测的阳性率,以PCR及血培养阴性、阳性患者血常规、TNF-α、IL-6、CRP、FPG、餐后2 h血糖(2 h PG)、Hb A1c的相关性。结果采用PCR和血培养检测阳性率和操作时间,组间比较差异有统计学意义(P<0.05)。16SrRNA基因检测及血培养方法阳性及阴性标本患者白细胞计数(WBC)、CRP、IL-6、TNF-α水平比较差异有统计学意义(P<0.05),且阳性患者明显高于阴性患者,16SrRNA基因检测及血培养方法阳性及阴性标本患者FPG、2 h PG比较差异有统计学意义(P<0.05),但Hb A1c组间比较无统计学意义(P>0.05)。两种检测方法分离病原菌以革兰阴性菌为主,革兰阳性菌低于革兰阴性菌,但是行血培养阴性患者中有部分患者PCR检测阳性。结论老年糖尿病烧伤患者应早期行血常规、TNF-α、IL-6、CRP检测、监测FPG、2 h PG变化,一旦有潜在脓毒症风险,行16SrRNA基因PCR检测,判断细菌特征,早期给予抗感染对症治疗。16SrRNA基因PCR检测应用于老年糖尿病烧伤脓毒症患者细菌感染特异性、快速性、敏感性均较高,操作时间短,不受临床应用抗生素的影响。     

定量PCR联合基因芯片检测腹水细菌16SrRNA诊断自发性细菌性腹膜炎

目的 探讨定量PCR联合基因芯片检测腹水细菌16SrRNA基因诊断自发性细菌性腹膜炎(SBP)的意义.方法 采用实时荧光定量PCR联合基因芯片检测76例临床疑似SBP肝病患者和6例对照的非感染性腹腔积液肝病患者腹水细菌16SrRNA基因,与腹水细菌培养同时比较.结果76份疑似SBP患者腹水标本中,定量PCR联合基因芯片检测阳性17份,阳性率为22.4%,其中革兰氏阳性菌8份、革兰氏阴性菌9份;腹水细菌培养阳性6份,阳性率为7.9%,均为革兰氏阴性菌,两种方法比较,x2=18.05,P＜0.01,差异有统计学意义.两种方法检测腹水细菌阳性的6份标本,菌株鉴定结果相一致.对照病例细菌检测结果呈阴性.结论 定量PCR联合基因芯片检测腹水细菌16SrRNA基因,较腹水细菌培养的敏感性和特异性高;不仅能作出快速诊断,还能确定SBP所感染的病原菌,具有实际应用价值.

Abstract：

Objective To evaluate the significance of determining ascitic bacterial16S rRNA by quantitative PCR combined with microarray (PCR-microarray) in the diagnosis of spontaneous bacterial peritonitis (SBP). Methods Ascitic bacterial 16SrRNA was determined by real time fluorescent quantitative PCR-microarray in 76 cases of suspected SBP and 6 cases of non-infectious ascites with chronic liver diseases.The results were compared with ascitic bacterial culture simultaneously. Results Of 76 ascitic samples, 17were detected bacteria positive by PCR-microarray, including 8 Grams positive(G+) and 9 Grams negative (G-), which was higher than that by bacterial culture which had only 6 ascitic samples detected positive (all G-); the positive rates were 22.4% vs 7.9%, respectively (P ＜ 0.01). The bacterial strains detected by both methods in 6 cases had a consistency with each other. No bacteria were detected in another 6 cases of noninfectious ascites with chronic liver diseases. Conclusions Determination of ascitic bacteria 16S rRNA by PCR-microarray has a higher specificity and sensitivity in the diagnosis of SBP as compared with the bacteria culture. Application of this novel method can not only accelerate SBP diagnosis but also stratify the different pathogens.     

链置换扩增技术在结核病诊断中的应用

目的探讨链置换扩增（SDA）技术检测结核分枝杆菌复合群（MTBC）的临床意义和可信性。方法应用SDA技术与荧光定量聚合酶链反应（FQ—PCR），直接检测了453份结核性样本，其中痰标本332份、胸腔积液78份、脑脊液43份。结果332份痰标本培养阳性131份，其中110株（涂阳88份）为MTBC，21株（涂阳20份）为非结核分枝杆菌。在110份证实有MTBC、21份证实有非结核分枝杆菌生长的样本中，SDA和FQ-PCR的敏感性分别为99．1％和95．2％，特异性分别为94．6％和95．2％。在311例结核分枝杆菌感染的患者中，SDA和FQ-PCR的阳性率分别为55．3％（172／311）和47.0％（146／311）。121份结核性胸腔积液、脑脊液样本中，分枝杆菌阳性20例，经鉴定其中19例为结核分枝杆菌，1例为非结核分枝杆菌，SDA和FQ—PCR检测的阳性率分别为43．4％（52／120）和33．4％（40／120）。SDA技术设立的内扩增质量控制（IAC）可确保检测结果的准确性。结论自动检测分析系统能快速、特异地检测临床样本中MTBC，设立IAC可提高结果的可信性。     

Neonatal sepsis in hospital born babies

Incidence of neonatal sepsis in a study carried out among hospital born babies was found to be 5.3 per cent significantly high (10.9%) amongst low birth weight compared to (3.1%) normal birth weight babies. Sepsis related mortality also exceeded significantly in low birth weight babies. Positive cultures were obtained in 36.7 per cent of babies with sepsis. The organisms identified were Staphylococcus pyogenes (40%), E. coli (27.5%), Klebsiella spp. (15%), Staphylococcus epidermidis (10%) and Enterobacter spp. (7.5%). Gram negative bacilli predominated in early onset (< 72 hrs. of life) and gram positive cocci in late onset. Mortality with early onset culture positive neonatal sepsis was significantly high compared to late onset. The bacterial isolates obtained were found to be resistant to routinely used antibiotics (penicillin, ampicillin and gentamycin). Third generation cephalosporins and aminoglycosides (netilmycin) were found to be effective in treatment of neonatal sepsis.     

Molecular diagnosis of infective endocarditis--a new Duke's criterion.

The molecular approach of PCR amplification of specific gene targets and universal loci for bacteria (16S rRNA) and fungi (18S, 28S and 5.8S rRNA) and subsequent sequencing was used to identify the possible causal microbial agent(s) in blood culture (47 patients) and heart valve material (30 patients) from patients with suspected infective endocarditis (IE). Culture and molecular results were analysed with respect to the patients' clinical background and the Duke Criteria. The findings demonstrated that: (i) all patients who were definite or possible cases were positive by PCR, even patients whose blood culture and valve material were culture-negative; and (ii) all patients who were rejected as having IE were also negative by PCR, with the exception of 1 patient who had bacteraemia from another source and 5 patients whose blood culture material was believed to contain an environmental contaminant. Direct molecular identification of the aetiological agents responsible for IE from blood culture material may enable specific treatment to commence at an earlier stage of the disease and hence reduce the need for valve replacement. Such a molecular approach may aid in the diagnosis of IE and should therefore be included as an additional major criterion in the Duke's classification scheme.     

Molecular diagnosis of infective endocarditis by real-time broad-range polymerase chain reaction (PCR) and sequencing directly from heart valve tissue

Traditionally, infective endocarditis (IE) has been microbiologically diagnosed by blood cultures or serology. However, conventional microbiologic methods do not always provide an etiologic diagnosis. We conducted the current study to evaluate the usefulness of a universal real-time polymerase chain reaction (PCR) of the 16S rRNA gene followed by sequencing for the diagnosis of IE in explanted heart valve tissue (HV) as part of the routine of a clinical microbiology laboratory, and to compare it with conventional culture of blood or HV. We prospectively analyzed 177 HV samples by universal PCR and sequencing: 48 were from 35 patients with definite IE and 129 were from 120 patients without IE. Specific PCR tests were used when necessary to confirm broad-range PCR results. For the 35 patients with IE, all of the HV samples except for 2 from the same patient gave positive PCR results. The microorganisms identified matched those isolated by blood culture in 31 cases. The other 3 patients had negative blood culture IE, but PCR made possible the detection of Tropheryma whipplei, Bartonella quintana, and Streptococcus gallolyticus. For the negative control group, universal PCR was completely negative in 123 of the 129 samples. Sensitivity, specificity, and negative and positive predictive values of this real-time PCR method were 96%, 95.3%, 98.4%, and 88.5%, respectively, for the diagnosis of IE, using the Duke criteria to define IE and using blood culture results to identify etiologic microorganisms. Conventional HV culture correlated poorly with blood cultures and molecular techniques, and frequently represented tissue contamination resulting from valve handling. Our universal PCR method has proved to be more sensitive, specific, and rapid than conventional culture methods, and should therefore be included as a new major Duke criterion for the diagnosis of IE. According to the results of the current study, this technique should be used to supplement blood and HV culture. Conventional HV cultures are frequently responsible for false-positive and false-negative results, and are not always useful to establish the etiology of IE.     

Molecular detection of bacterial contamination in gnotobiotic rodent units

Gnotobiotic rodents provide an important technique to study the functional roles of commensal bacteria in host physiology and pathophysiology. To ensure sterility, these animals must be screened frequently for contamination. The traditional screening approaches of culturing and Gram staining feces have inherent limitations, as many bacteria are uncultivable and fecal Gram stains are difficult to interpret. Thus, we developed and validated molecular methods to definitively detect and identify contamination in germ-free (GF) and selectively colonized animals. Fresh fecal pellets were collected from rodents housed in GF isolators, spontaneously contaminated ex-GF isolators, selectively colonized isolators and specific pathogen-free (SPF) conditions. DNA isolated from mouse and rat fecal samples was amplified by polymerase chain reaction (PCR) and subjected to quantitative PCR (qPCR) using universal primers that amplify the 16S rRNA gene from all bacterial groups. PCR products were sequenced to identify contaminating bacterial species. Random amplification of polymorphic DNA (RAPD) PCR profiles verified bacterial inoculation of selectively colonized animals. These PCR techniques more accurately detected and identified GF isolator contamination than current standard approaches. These molecular techniques can be utilized to more definitively screen GF and selectively colonized animals for bacterial contamination when Gram stain and/or culture results are un-interpretable or inconsistent.     

Detection of ascitic fluid infections in patients with liver cirrhosis and ascites

Background and study aimsAscitic fluid infections (AFIs) are the frequent complications of advanced liver disease. Bacterial translocation is considered a key step in the pathogenesis of gut-derived bacterial infections; mainly spontaneous bacterial peritonitis (SBP) in cirrhotic patients. Bacterial DNA (bactDNA) in ascitic fluid and serum has been suggested as a surrogate marker for bacterial translocation. We attempted at the isolation and identification of bacteria in ascitic fluid in cirrhotic patients and the assessment of polymerase chain reaction (PCR) in ascitic fluid and serum.Patients and methodsFifty cirrhotic patients having ascites with no signs of infection were included. Ascitic fluid cultures were obtained from patients. Ascitic fluid and serum were subjected to DNA extraction and PCR for the universal amplification of a region of the 16S ribosomal RNA (16S rRNA) gene to detect bactDNA.ResultsBacteria were isolated from 9 (18%) of the ascitic fluid samples, and were mainly Gram-positive bacteria. BactDNA was detected simultaneously in the ascitic fluid and serum of 17 (34%) patients and in the ascitic fluid of only 2 patients. In a single patient with positive ascitic fluid culture no bactDNA was detected in ascitic fluid or serum. By considering AFIs as a positive ascitic fluid culture and/or the presence of bactDNA in the ascitic fluid and/or serum, ascitic fluid culture could detect 9 out of 20 patients with AFIs (45%), PCR of ascitic fluid could detect 19 out of 20 (95%) while PCR of serum could detect 17 out of 20 (85%). In 10 patients with culture negative non-neutrocytic ascites (CNNNA) bactDNA could be detected in serum and ascitic fluid.ConclusionAFI can be caused by Gram positive as well as Gram negative organisms. A substantial percentage of cases with CNNNA show bactDNA in serum and ascitic fluid. PCR of ascitic fluid should, therefore, be used in the diagnostic workup of suspected cases of ascitic fluid infections.     

rRNA扩增方法对结核分枝杆菌临床诊断作用的评估

目的 评价rRNA扩增方法 在临床应用的效果。 方法 选取到北京胸科医院、山东省胸科医院、河南疾控中心结核病防治所3个机构就诊的肺结核可疑症状者及健康志愿者的痰标本为研究对象,共纳入551例痰标本,对每例痰标本均进行涂片显微镜检查、罗氏培养、rRNA扩增试验以及实时荧光定量PCR。与罗氏培养方法 比较分析rRNA扩增方法 的敏感性、特异性、阳性预测值和阴性预测值以及与实时荧光定量PCR扩增方法 的一致性。 结果rRNA扩增方法 的敏感性为98.5%,特异性为95.0%,阳性预测值为95.0%,阴性预测值为98.5%,rRNA扩增方法 在涂阳标本和涂阴标本间的敏感度和特异度差异均有统计学意义（χ²=9.60,P=0.002;χ²=79.80,P<0.01）。与PCR检测结果的一致性为93.8%,涂阳和涂阴标本中2种方法 的一致性差异有统计学意义（χ²=4.45,P=0.035）。 结论 rRNA扩增方法 的敏感度和特异度均较好,且能明显缩短诊断时间,该方法 是一种较有前景的结核病实验室诊断方法 。     

Broad-range real time PCR and DNA sequencing for the diagnosis of bacterial meningitis

Rapid aetiological diagnosis of bacterial meningitis is crucial for the early targeting of antimicrobial and adjuvant therapy. Broad-range polymerase chain reaction (PCR) targeting the 16S rRNA gene allows aetiological diagnosis of bacterial meningitis when applied to cerebrospinal fluid (CSF). We assessed the additional diagnostic effect of applying a novel broad-range real time PCR and subsequent DNA sequencing to culture, microscopy, and broad-range conventional PCR on CSF in patients with suspected bacterial meningitis. Broad-range conventional PCR and broad-range real time PCR with subsequent DNA sequencing were applied to 206 CSF specimens collected consecutively from 203 patients aged 6 d to 86 y. Patients' charts were reviewed for clinical information. 17 pathogens were identified by PCR and DNA sequencing or culture. Three specimens were negative by culture but positive by broad-range real time PCR. Three specimens were positive by culture but negative by broad-range real time PCR. Compared with culture, the sensitivity of broad-range real time PCR was 86%, and the specificity 98%. Conventional PCR resulted in a sensitivity of 64% and specificity of 98%. Broad-range real time PCR was generally comparable to culture of CSF and may be a useful supplement, particularly when antimicrobial therapy has been administered. Broad-range real time PCR was more sensitive than broad-range conventional PCR and microscopy.