MALDI-TOF MS技术在侵袭性丝状真菌快速鉴定中的应用

探索不同培养基、不同培养时间和不同蛋白质提取方法对侵袭性丝状真菌质谱鉴定准确率的影响, 旨在提高基质辅助激光解析电离飞行时间质谱技术鉴定侵袭性丝状真菌的准确率。

采用分子生物学方法为金标准, 同时运用基质辅助激光解析电离飞行时间质谱技术对所收集临床丝状真菌进行鉴定。根据分子生物学的鉴定结果, 去除VITEK-MS v3.0数据库中没有的菌株, 其余菌株接种在沙氏葡萄糖琼脂(SDA)、马铃薯葡萄糖琼脂(PDA)和察氏培养基(CA)3种不同的培养基中, 采用2种不同的蛋白质提取方法(甲酸乙腈法和磁珠法), 获得了不同培养时间点(2、3、5、7和9 d)的特异性质谱指纹图谱。

不同丝状真菌蛋白质提取方法进行比较, 甲酸乙腈法总鉴定准确率为79.8%, 磁珠法总鉴定准确率为77.5%, 2种丝状真菌蛋白质提取方法的质谱鉴定准确率差异无统计学意义(χ²=1.040, P=0.308)。不同培养基进行比较, SDA培养基总鉴定准确率为90.7%, PDA培养基总鉴定准确率为81.4%, CA培养基总鉴定准确率为67.4%, 3种不同培养基的丝状真菌质谱鉴定准确率差异有统计学意义(χ²=36.609, P < 0.001), 其中使用SDA培养基鉴定准确率最高, 使用CA培养基鉴定准确率最低(SDA vs PDA, χ²=7.748, P=0.005;SDA vs CA, χ²=35.131, P < 0.001;PDA vs CA, χ²=10.994, P=0.001)。不同培养时间进行比较, 丝状真菌培养2、3、5、7和9 d后的质谱总鉴定准确率分别为64.3%、88.4%、89.1%、79.1%和78.3%, 不同培养时间的质谱鉴定准确率差异有统计学意义(χ²=32.274, P < 0.001)。培养3 d和培养5 d的质谱鉴定准确率优于培养7 d和培养9 d的质谱鉴定准确率, 培养2 d的质谱鉴定准确率明显低于其他时间(3 d vs 5 d, χ²=0.039, P=0.844;7 d vs 9 d, χ²=0.023, P=0.879;3 d vs 7 d, χ²=4.095, P=0.043;2 d vs 9 d, χ²=6.139, P=0.013)。

侵袭性丝状真菌使用SDA培养基培养3 d, 运用甲酸乙腈法提取蛋白质进行质谱鉴定最理想。

     

厄贝沙坦片联合益生菌对高血压伴肥胖患者的疗效及对肠道菌群的调控作用

探讨厄贝沙坦片联合益生菌对高血压伴肥胖患者的疗效及对肠道菌群的调控作用。

选取2021年10月—2022年10月本院400例高血压伴肥胖患者。随机法分组：对照组（200例）给予厄贝沙坦治疗,研究组（200例）在对照组基础上联用益生菌。比较两组治疗前后舒张压（DBP）、收缩压（SBP）、C反应蛋白（CRP）、白细胞介素（IL）-6水平和肠道菌群变化,统计治疗期间不良反应发生情况。

治疗后,研究组DBP、SBP、CRP和IL-6水平均低于对照组（P＜0.05）。治疗后,研究组乳杆菌、拟杆菌及双歧杆菌水平高于对照组,肠杆菌及肠球菌水平低于对照组（P＜0.05）。研究组不良反应发生率为3.0%,对照组为4.5%,差异无统计学意义（χ² = 0.623,P = 0.430）。

厄贝沙坦联合益生菌治疗高血压伴肥胖可提升患者血压控制效果,减轻炎症反应,改善肠道紊乱菌群,且不良反应少。

     

经颅直流电刺激联合益生菌对脑卒中后认知功能障碍患者的作用

探讨经颅直流电刺激（tDCS）联合益生菌对脑卒中后认知功能障碍（PSCI）患者认知功能和肠道菌群的影响。

按随机数字表法将2020年6月至2021年6月我院收治的150例PSCI患者分为对照组和观察组,各75例。对照组患者给予tDCS治疗,观察组患者给予tDCS联合益生菌治疗。采用蒙特利尔认知功能评估量表（MoCA）和简易智力状态检查量表（MMSE）评估患者治疗前后的认知功能。采用荧光定量PCR法检测患者治疗前后肠道菌群数量变化。比较两组患者认知功能、肠道菌群、临床疗效以及不良反应发生情况。

治疗后,观察组患者肠道双歧杆菌和乳杆菌数量较治疗前显著升高,且双歧杆菌和乳杆菌数量显著高于对照组;而大肠埃希菌和肠球菌数量较治疗前显著降低,且大肠埃希菌和肠球菌数量显著低于对照组（均P＜0.05）。治疗后,两组患者MoCA和MMSE评分均升高,且观察组高于对照组（均P＜0.05）。观察组患者临床总有效率显著高于对照组（90.67% vs 78.67%,χ² = 12.482,P＜0.001）。对照组和观察组患者的不良反应发生情况比较差异无统计学意义（χ² = 0.150,P = 0.699）。

tDCS联合益生菌治疗能够有效改善PSCI患者的认知功能和肠道菌群状态,且不良反应少,安全系数高,值得临床推广。

     

加味甘麦大枣汤对气血两虚型产后抑郁症患者肠道菌群和血清5-HT的影响

观察加味甘麦大枣汤对气血两虚型产后抑郁症（PPD）患者肠道菌群和血清5-羟色胺（5-HT）水平的影响。

选取2022年10月至2023年8月就诊于甘州区妇幼保健院且符合纳入标准的气血亏虚型产后抑郁症患者60例作为研究对象,按其就诊顺序随机分为观察组、对照组,每组各30例。对照组给予西医常规治疗,观察组在西医常规治疗基础上联合加味甘麦大枣汤治疗,疗程4周。分别于治疗前和治疗后采用中医证候积分和汉密尔顿抑郁量表（HAMD）评价治疗疗效。通过高通量测序法检测两组患者治疗前后粪便中的菌群多样性和各分类学水平分布。采用酶联免疫吸附测定（ELISA）检测两组患者血清5-HT水平。

与本组治疗前比较,治疗后两组患者中医证候积分和HAMD评分均明显下降,差异具有统计学意义（P＜0.05）。治疗后,与对照组比较,观察组的中医证候积分和HAMD评分显著下降（P＜0.05）。观察组临床有效率为96.7%,高于对照组的76.7%（χ²=8.967,P=0.030）。观察组肠道菌群在治疗后的Simpson指数和Shannon指数较治疗前升高（P＜0.05）,拟杆菌门（Bacteroidetes）、瘤胃菌科（Ruminococcaceae）、拟杆菌属（Bacteroides）和双歧杆菌属（Bifidobacterium）相对丰度显著高于对照组（P＜0.05）,变形菌门（Proteobacteria）、肠杆菌科（Enterobacteriaceae）、粪球菌属（Coprococcus）和志贺菌属（Shigella）相对丰度显著低于对照组（P＜0.05）。两组患者血清5-HT水平治疗后均明显升高（P＜0.05）,观察组5-HT水平升高的更明显（P=0.010）。

加味甘麦大枣汤可辅助临床治疗改善PPD患者的临床症状,调节患者肠道菌群,提高血清5-HT水平。

     

双歧杆菌乳杆菌三联活菌片对幽门螺杆菌根治后消化道不良反应的改善作用

探讨加用双歧杆菌乳杆菌三联活菌片对临床四联疗法根除幽门螺杆菌(H.pylori)过程中患者消化道不良反应的改善作用。

选择2019年1月至2020年1月在昆明医科大学第二附属医院门诊就诊的100例H.pylori感染患者, 分为益生菌组和正常组, 各50例。全部患者通过四联疗法根除H.pylori后, 益生菌组患者继续加用双歧杆菌乳杆菌三联活菌片治疗, 对结果进行分析。

2组患者使用含有铋剂的四联疗法根除H.pylori后效果满意。益生菌组患者不良反应发生率低于正常组(25.5% vs 47.5%;χ²=11.023, P=0.001)。

益生菌可减轻四联疗法根除H.pylori后消化道不良反应, 根除H.pylori过程中加用益生菌的时机尚待进一步探讨。

     

高危型人乳头瘤病毒感染与阴道微生态及宫颈局部细胞免疫的相关性

探讨高危型人乳头瘤病毒(HR-HPV)感染与阴道微生态及宫颈局部细胞免疫的相关性, 为该类患者的治疗提供参考。

选择2018年1月至2020年1月我院收治的126例HPV感染者, 依据感染病毒类型分为非HR-HPV感染组(n=69)和HR-HPV感染组(n=57), 并纳入同期来我院体检的50名健康体检者作为对照组。分析HPV感染类型、患者阴道微生态状况及宫颈局部组织T淋巴细胞亚群水平。

患者中复合型与单一型HR-HPV感染率分别为15.8%、84.2%, 差异有统计学意义(χ²=5.324, P=0.047)。对照组、非HR-HPV感染组和HR-HPV感染组对象阴道微生态失调率分别为22.0%、34.8%、45.6%, 其中对照组对象阴道微生态失调率最低(χ²=5.638, P=0.048)。非HR-HPV感染组和HR-HPV感染组患者阴道病原菌检出率高于对照组(均P < 0.05)。非HR-HPV感染组和HR-HPV感染组患者宫颈局部T淋巴细胞亚群水平低于对照组(均P < 0.05)。

HPV33、16、18型为HPV感染主要类型, 会加重阴道微生态紊乱程度, 影响局部细胞免疫功能。检测患者HPV感染情况和宫颈局部细胞免疫功能可为患者临床治疗提供有效数据, 有利于治疗措施的制定。

     

支气管镜下冷冻联合镜下给药治疗溃疡型支气管结核的临床效果

探讨支气管镜下冷冻联合局部镜下注药对溃疡型支气管结核的临床效果,为该病的治疗提供参考。

选取2019年1月至2021年1月大连市公共卫生临床中心结核科收治的溃疡坏死型支气管结核患者80例,根据治疗方案分为对照组（n = 40,给予常规规范抗结核治疗）和研究组（n = 40,在对照组基础上给予支气管镜下冷冻联合局部注药治疗）。观察两组患者治疗4周后疗效、痰菌转阴时间及治疗前和治疗4周后第1秒用力呼气量（FEV1）、白细胞介素-10（IL-10）、C反应蛋白（CRP）、血沉（ESR）水平及治疗期间不良反应情况。

研究组患者总有效率高于对照组,痰菌转阴时间短于对照组（均P＜0.05）,但两组患者不良反应发生率比较差异无统计学意义（15.0% vs 12.5%,P＞0.05）。两组患者治疗后FEV1水平较治疗前上升,IL-10、CRP、ESR水平较治疗前下降,研究组患者上述指标水平治疗前后差值大于对照组（均P＜0.05）。

支气管镜下冷冻联合局部镜下注药对溃疡型支气管结核的疗效较好,可抑制机体炎症介质的释放,缩短痰菌转阴时间,安全性较高。

     

基于高通量测序分析哮喘小鼠呼吸道菌群的变化

通过高通量测序分析哮喘模型小鼠呼吸道菌群的变化情况。

将12只SPF级BALB/c雄性小鼠随机分为对照组和模型组, 每组6只。采用卵清蛋白致敏方法建立哮喘小鼠模型后, 进行支气管组织切片病理学观察, ELISA法检测血清IgE水平, 测定肺指数, 采集咽拭子后提取DNA行高通量测序分析。

与对照组比较, 模型组小鼠血清IgE水平明显升高(P < 0.05), 肺指数明显上升(P < 0.05), 可见支气管上皮粘膜有水肿, 少量淋巴细胞浸润, 平滑肌增生。模型组小鼠呼吸道菌群与对照组比较, 菌种丰度升高, 厚壁菌门较对照组减少(P < 0.05), 放线菌门和变形菌门增多(P < 0.05), 菌群结构有明显差异。

哮喘小鼠存在呼吸道微生态菌群失衡。

     

急性脑卒中患者肠道菌群研究

通过粪便标本检测急性脑卒中患者肠道菌群变化情况, 探讨急性脑卒中患者肠道菌群结构。

通过高通量二代测序技术对10例健康者(对照组)及10例急性脑卒中患者(疾病组)粪便样本进行菌群结构测序分析。

与对照组比较, 急性脑卒中患者粪便样本中物种OTU信息量显著增加(P < 0.01), 菌群多样性指数(Shannon)和物种均一度指数(Evenness)也有所增加但差异无统计学意义(均P > 0.05)。门水平上, 疾病组患者肠道Bacteroidetes数量较对照组显著增加, Firmicutes数量显著减少(均P < 0.05)。属水平上, 疾病组患者肠道Bacteroides、Bilophila、Butyricimonas比例较对照组显著升高, 而Collinsella、Coprococcus、Clostridium等比例较对照组显著降低(均P < 0.05)。

急性脑卒中患者肠道菌群结构与健康人存在显著差异。

     

Caspase-4非经典炎症小体对问号钩体诱导THP-1细胞炎症因子分泌水平的影响

了解Caspase-4非经典炎症小体在问号钩体诱导THP-1细胞炎性细胞因子分泌过程中的作用。

采用问号钩体感染THP-1细胞(预先经佛波酯刺激分化为巨噬细胞)建立细胞模型, 用实时荧光PCR扩增检测caspase-4、IL-18、IL-1β和IL-1αmRNA水平, Western blotting检测Caspase-4蛋白表达, ELISA定量检测细胞上清中IL-18、IL-1β和IL-1α分泌情况。

实时荧光PCR和Western blotting显示, 与未感染细胞比较, THP-1细胞Caspase-4 mRNA及蛋白表达水平均升高(t=46.03、29.36, 均P < 0.05), Caspase-4 siRNA转染后, Caspase-4 mRNA及蛋白表达水平显著下降(t=32.48、30.77, 均P < 0.01);钩体感染后, IL-18、IL-1β和IL-1αmRNA水平显著升高(t=25.70、26.13、19.94, 均P < 0.05), 在Caspase-4特异性阻断后显著下降(t=11.55、44.68、15.68, 均P < 0.05);ELISA检测显示, 钩体感染后, IL-18、IL-1β和IL-1α分泌均显著升高(t=50.24、34.17、25.18, 均P < 0.05), 且能被Caspase-4特异性阻断剂有效抑制(t=42.00、17.07、5.03, 均P < 0.05)。

Caspase-4非经典炎症小体参与介导问号钩体诱导人单核-巨噬细胞炎性细胞因子IL-18、IL-1β和IL-1α的分泌。

     

An efficient alternative marker for specific identification of Mycobacterium tuberculosis

Rapid and accurate identification of mycobacteria to the species level is important to provide epidemiological information and to guide the appropriate treatment, especially identification of the Mycobacterium tuberculosis (MTB) which is the leading pathogen causing tuberculosis. The genetic marker named as Mycobacterium tuberculosis specific sequence 90 (mtss90) was screened by a bioinformatics software and verified by a series of experiments. To test its specificity, 266 strains of microorganisms and human cells were used for the mtss90 conventional PCR method. Moreover, the efficiency of mtss90 was evaluated by comparing 16S rDNA (Mycobacterium genus-specific), IS6110 (specific identification of MTB complex), mtp40 (MTB-specific) and PNB/TCH method (traditional bacteriology testing) in Mycobacterium strains. All MTB isolates were mtss90 positive. No amplification was observed from any other tested strains with M. microti as an exception. Compared with the traditional PNB/TCH method, the coincidence rate was 99.1 % (233/235). All of the mtss90 positive strains were IS6110 and 16S rDNA positive, indicating a 100 % coincidence rate (216/216) between mtss90 and these two genetic markers. Additionally, mtss90 had a better specificity than mtp40 in the identification of MTB. Lastly, a real-time PCR diagnostic assay was developed for the rapid identification of MTB. In conclusion, mtss90 may be an efficient alternative marker for species-specific identification of MTB and could be used for the diagnosis of tuberculosis combined with other genetic markers.     

Evaluation of a Simple in-House Test to Presumptively Differentiate Mycobacterium tuberculosis Complex from Nontuberculous Mycobacteria by Detection of p-Nitrobenzoic Acid Metabolites

The timely differentiation of Mycobacterium tuberculosis complex (MTC) and non-tubercular mycobacterium (NTM) species is urgently needed in patient care since the routine laboratory method is time consuming and cumbersome. An easy and cheap method which can successfully distinguish MTC from NTM was established and evaluated. 38 mycobacterial type and reference strains and 65 clinical isolates representing 10 species of mycobacterium were included in this study. Metabolites of p-nitrobenzoic acid (PNB) reduction were identified using liquid chromatography and tandem mass spectrometry (LC/MS/MS). A spectrophotometric method was developed to detect these metabolites, which was evaluated on a number of MTC and NTM species. All of the tested NTM species and strains reduced PNB to p-aminobenzoic acid (PABA), while none of the MTC strains showed a similar activity. Spectrophotometric detection of PABA had 100% sensitivity and specificity for MTC and NTM differentiation among the type strains and the clinical isolates tested. PABA was identified as one of the metabolites of PNB reduction. All the tested NTM species metabolized PNB to PABA whereas the MTC members lacked this activity. A simple, specific and cost-effective method based on PABA production was established in order to discriminate MTC from NTM from cultured organisms.     

Diagnostic Accuracy and Turnaround Time of the Xpert MTB/RIF Assay in Routine Clinical Practice

The Xpert MTB/RIF assay was introduced for timely and accurate detection of tuberculosis (TB). The aim of this study was to determine the diagnostic accuracy and turnaround time (TAT) of Xpert MTB/RIF assay in clinical practice in South Korea. We retrospectively reviewed the medical records of patients in whom Xpert MTB/RIF assay using sputum were requested. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the diagnosis of pulmonary tuberculosis (PTB) and detection of rifampicin resistance were calculated. In addition, TAT of Xpert MTB/RIF assay was compared with those of other tests. Total 681 patients in whom Xpert MTB/RIF assay was requested were included in the analysis. The sensitivity, specificity, PPV and NPV of Xpert MTB/RIF assay for diagnosis of PTB were 79.5% (124/156), 100.0% (505/505), 100.0% (124/124) and 94.0% (505/537), respectively. Those for the detection of rifampicin resistance were 57.1% (8/14), 100.0% (113/113), 100.0% (8/8) and 94.9% (113/119), respectively. The median TAT of Xpert MTB/RIF assay to the report of results and results confirmed by physicians in outpatient settings were 0 (0–1) and 6 (3–7) days, respectively. Median time to treatment after initial evaluation was 7 (4–9) days in patients with Xpert MTB/RIF assay, but was 21 (7–33.5) days in patients without Xpert MTB/RIF assay. Xpert MTB/RIF assay showed acceptable sensitivity and excellent specificity for the diagnosis of PTB and detection of rifampicin resistance in areas with intermediate TB burden. Additionally, the assay decreased time to the initiation of anti-TB drugs through shorter TAT.     

Validation of Biomarkers for Distinguishing Mycobacterium tuberculosis from Non-Tuberculous Mycobacteria Using Gas Chromatography?Mass Spectrometry and Chemometrics

Tuberculosis (TB) remains a major international health problem. Rapid differentiation of Mycobacterium tuberculosis complex (MTB) from non-tuberculous mycobacteria (NTM) is critical for decisions regarding patient management and choice of therapeutic regimen. Recently we developed a 20-compound model to distinguish between MTB and NTM. It is based on thermally assisted hydrolysis and methylation gas chromatography-mass spectrometry and partial least square discriminant analysis. Here we report the validation of this model with two independent sample sets, one consisting of 39 MTB and 17 NTM isolates from the Netherlands, the other comprising 103 isolates (91 MTB and 12 NTM) from Stellenbosch, Cape Town, South Africa. All the MTB strains in the 56 Dutch samples were correctly identified and the model had a sensitivity of 100% and a specificity of 94%. For the South African samples the model had a sensitivity of 88% and specificity of 100%. Based on our model, we have developed a new decision-tree that allows the differentiation of MTB from NTM with 100% accuracy. Encouraged by these findings we will proceed with the development of a simple, rapid, affordable, high-throughput test to identify MTB directly in sputum.     

Performance Assessment of the BluePoint MycoID Plus Kit for Identification of Mycobacterium tuberculosis,Including Rifampin- and Isoniazid-resistant Isolates,and Nontuberculous Mycobacteria

The performance of the BluePoint MycoID plus kit (Bio Concept Corporation, Taichung, Taiwan), which was designed to simultaneously detect Mycobacterium tuberculosis (MTB), rifampin- and isoniazid-resistant MTB, and nontuberculous mycobacteria (NTM) was first evaluated with 950 consecutive positive cultures in Mycobacterium Growth Indicator Tube (MGIT) system (BACTEC, MGIT 960 system, Becton-Dickinson, Sparks) from clinical respiratory specimens. The discrepant results between kit and culture-based identification were finally assessed by 16S rRNA gene sequencing and clinical diagnosis. The accuracy rate of this kit for identification of all Mycobacterium species was 96.3% (905/940). For MTB identification, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the kit were 99.7%, 99.3%, 99.0% and 99.8%, respectively. For rifampicin-resistant MTB identification, the sensitivity, specificity, PPV, and NPV of the kit were 100.0%, 99.4%, 91.3%, and 100.0%, respectively, while the corresponding values of isoniazid-resistant MTB identification were 82.6%, 99.4%, 95.0%, and 97.6%, respectively. In identifying specific NTM species, the kit correctly identified 99.3% of M. abscessus (147/148) complex, 100% of M. fortuitum (32/32), M. gordonae (38/38), M. avium (39/39), M. intracellulare (90/90), M. kansasii (36/36), and M. avium complex species other than M. avium and M. intracellulare (94/94). In conclusions, the diagnostic value of the BluePoint MycoID plus kit was superior to culture method for recoveries and identification of NTM to species level. In addition, the diagnostic accuracy of BluePoint MycoID plus kit in MTB identification was similar to conventional culture method with high accuracy rate of rifampicin-resistant M. tuberculosis identification.     

甲胎蛋白胶体金检测试剂的的研制

为建立一种快速,简易胶体金免疫层析法（GICA）用于检测血清中的甲胎蛋白（AFP）,将AFP单克隆抗体分别标记胶体金并包被硝酸纤维素膜,制成免疫检测层析试剂,血清中的AFP与金标记抗体结合,沿着硝酸纤维素膜移动,与膜上的固相抗体结合可形成肉眼可见的紫红色线条,此检测试剂对本室自制AFP参比品进行了检测,灵敏度达20ng/ml;对甘肃省肿瘤医院的176份癌症患者血清进行了检测。结果与ELISA法相一致,本法检测AFP快速,简便,结果准确,具有推广价值。     

土拉弗朗西斯菌胶体金免疫层析快速检测方法的建立

目的建立胶体金免疫层析技术快速定量检测土拉弗朗西斯菌。方法利用胶体金标记和双抗体夹心免疫层析技术,建立土拉弗朗西斯菌的快速检测方法,评价其特异性和敏感性,并拟合检测曲线进行定量检测。在面粉、饼干、果冻、梨汁等食品样品中添加土拉弗朗西斯菌的FopA蛋白模拟污染样品,评价该方法对固体、半固体、液体等食品样品的检测能力。结果该法可在10min内完成定性和定量检测,灵敏度为750ng／ml,线性范围750～24000ng/ml、回收率为56．7％-89．2％。结论所建立的检测土拉弗朗西斯菌的胶体金免疫层析方法,能快速、灵敏、特异、准确地检测样品中的土拉弗朗西斯菌,适用于现场快速检测。     

Development of a real-time PCR-based method for rapid differential identification of Mycobacterium species

AIMS: To develop a real-time PCR method for rapid differential identification of many clinically important mycobacteria to the species level. METHODS AND RESULTS: Eighteen Mycobacterium species that are considered clinically important were targeted for the identification. One primer pair and 21 pairs of hybridization probes (HybProbes) specific for the genus, species or complex were designed based on the rpoB gene sequences of mycobacteria. Twenty-five different Mycobacterium reference species were tested. In a single round of real-time PCR, all the nontuberculous mycobacteria (NTM) species tested were identified at the genus level and 16 of the 18 targeted species were differentially identified to the species or complex level during the amplification cycles; subsequent melting curve analysis allowed the specific identification of all the target species at the species or complex level without cross-reactivity with the other species. CONCLUSIONS: The developed real-time PCR assay rapidly identifies the NTM at the genus level and 18 clinically important Mycobacterium species at the species or complex level. SIGNIFICANCE AND IMPACT OF THE STUDY: This real-time PCR assay provides a useful tool for the rapid differentiation of most clinically important Mycobacterium species.     

委内瑞拉马脑炎病毒重组抗原的制备纯化及其胶体金免疫层析检测方法的建立

目的:表达委内瑞拉马脑炎病毒E2重组蛋白,并结合胶体金免疫层析技术建立一种简便检测委内瑞拉马脑炎病毒特异性抗体的方法。方法:利用已经构建的表达E2抗原的工程菌, 用IPTG诱导, 表达蛋白主要以包涵体的形式存在。通过一系列条件的变性、复性、透析,所制抗原用以包被硝酸纤维素膜,利用胶体金标记和免疫层析技术,建立委内瑞拉马脑炎病毒快速检测方法。对该方法的敏感性、特异性和稳定性作出评价。结果:重组工程菌可表达分子质量为 40 kDa的目的蛋白, 纯化后的蛋白质经SDS-PAGE显示纯度达95%以上。建立的检测方法可在20 min内完成检测。对症状相似及近缘的其他病毒进行检测,均无非特异反应。试纸条在37℃下保存2周,检测结果不变。该方法与R&;D公司商品化的ELISA试剂盒灵敏度检测无明显差异;对92份阴性血清进行检测,两种检测方法的符合率为96.7%。结论:重组委内瑞拉马脑炎病毒蛋白产生的包涵体变性复性后生具有良好的重复性和稳定性, 可作为委内瑞拉马脑炎病毒多种检测方法的抗原原料。胶体金免疫层析法具有快速、灵敏、特异、稳定的特点,适用于现场检测。