基于蛋白L染色方法的流式细胞术检测嵌合抗原受体的表达

目的探讨以蛋白L(protein L)为基础的流式细胞染色方法用于检测患者T细胞表面嵌合抗原受体(chimeric antigen receptor,CAR)表达的技术可行性。方法单采2例CD19阳性淋巴瘤患者外周血单个核细胞(peripheral blood mononuclear cells,PBMCs),磁珠分选法获得T细胞,加入靶向CD19、CD22的CAR逆转录病毒,转染、诱导培养成CAR-T细胞。通过生物素化蛋白L(biotinylated protein L)-亲合素(streptavidin-PE)系统对CAR-T细胞表面的单链抗体片段(single-chain variable fragment,sc Fv)进行标记,用流式细胞术检测活化培养的T细胞中CD19-CAR、CD22-CAR表达阳性T细胞的比例,以常规Anti-Ig G染色的流式细胞检测方法作为对照组,进行结果对比。结果 sc Fv-biotinylated protein L-streptavidin-PE-流式细胞染色方法能够检测到CAR-T细胞表面CAR表达。2例患者蛋白L实验组和Anti-Ig G对照组CD19-CAR-T细胞比例分别为71.6%vs64.2%和49.3%vs 43.8%;CD22-CAR-T细胞比例分别为53.1%vs 46.3%和56.5%vs 64.0%,测定结果相似。结论以蛋白L为基础的染色方法可以作为流式细胞术检测CAR-T细胞的常规染色方法。     

核基质蛋白22及尿膀胱癌抗原在膀胱移行细胞癌诊断中的价值

目的 探讨核基质蛋白22(NMP22)及尿膀胱癌抗原(UBC)的检测用于早期诊断膀胱移行细胞癌(BTCC)的可行性及影响因素.方法 膀胱移行细胞癌患者60例,泌尿系良性疾病患者25例,健康对照组20名,采用酶联免疫(ELISA)方法检测尿NMP22及UBC.膀胱镜检查前取尿样分别进行NMP22、UBC和脱落细胞学检测,分析比较3种方法的敏感性、特异性、阳性和阴性预测价值.结果 以大于正常对照组尿液NMP22水平上限10 U/ml为NMP22阳性界值,以12μg/L为UBC诊断膀胱移行细胞癌的临界值时,NMP22和UBC诊断膀胱移行细胞癌的敏感性分别为88.3%和86.7%,与脱落细胞学(40.0%)比较,差异均有统计学意义(P均<0.01),3种方法诊断膀胱移行细胞癌的特异性分别为80.0%、84.0%和92.0%,阳性预测值分别为91.4%、92.9%和92.3%,阴性预测值分别为74.1%、72.4%和38.9%.结论 尿NMP22和UBC检测技术简单,有较高的敏感性和特异性,可作为早期诊断膀胱移行细胞癌的肿瘤标记物.     

核糖体蛋白L6对K562/A02细胞耐药性及凋亡的影响

本研究观察核糖体蛋白L6（RPL6）基因表达的改变对白血病细胞耐药性的作用及其可能的机制。通过RT-PCR方法获得RPL6 cDNA序列,用真核表达载体pcDNA3.1（＋）分别构建正向插入和反向插入的RPL6 cDNA重组质粒。以脂质体将正义RPL6 cDNA真核表达质粒转染K562细胞,将反义RPL6 cDNA真核表达质粒转染K562/AO2细胞。以MTT、流式细胞术和荧光分光光度计观察RPL6对化疗药物耐药性、凋亡和caspase-3的作用。结果表明：转染正义RPL6 cDNA真核表达质粒后,K562细胞对阿霉素的耐药性增强到原来的325%,凋亡和caspase-3活性明显降低（P〈0.005）;转染反义RPL6 cDNA真核表达质粒后,K562/A02细胞对阿霉素的耐药性降低原来的38%;凋亡和caspase-3活性明显增加（P〈0.005）。结论：RPL6基因过表达通过改变药物诱导的凋亡在K562/A02细胞耐药性的形成中起重要作用。     

Evaluation of a new multiplex polymerase chain reaction assay STDFinder for the simultaneous detection of 7 sexually transmitted disease pathogens

We evaluated a new multiplex polymerase chain reaction (mPCR), “STDFinder assay”, a novel multiplex ligation-dependent probe amplification (MLPA) assay for the simultaneous detection of 7 clinically relevant pathogens of STDs, i.e., Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, Mycoplasma genitalium, Treponema pallidum, and herpes simplex virus type 1 and 2 (HSV-1 and HSV-2). An internal amplification control was included in the mPCR reaction. The limits of detection for the STDFinder assay varied among the 7 target organisms from 1 to 20 copies per MLPA assay. There were no cross-reactions among any of the probes. Two hundred and forty-two vaginal swabs and an additional 80 specimens with known results for N. gonorrhoeae and C. trachomatis, obtained from infertile women seen at an infertility research clinic at the Kigali Teaching Hospital in Rwanda, were tested by STDFinder assay and the results were confirmed by single real-time PCR using different species-specific targets. Compared to the reference standard, the STDFinder assay showed specificities and sensitivities of 100% and 100%, respectively, for N. gonorrhoeae, C. trachomatis, and M. genitalium; 90.2% and 100%, respectively, for Trichomonas vaginalis; and 96.1% and 100%, respectively, for HSV-2. No specimen was found to be positive for HSV-1 by either the STDFinder assay or the comparator method. Similarly, the sensitivity for Treponema pallidum could not be calculated due to the absence of any Treponema pallidum-positive samples. In conclusion, the STDFinder assays have comparable clinical sensitivity to the conventional mono and duplex real-time PCR assay and are suitable for the routine detection of a broad spectrum of these STDs at relatively low cost due to multiplexing.     

2型糖尿病患者尿白蛋白/肌酐排泄率与血管内皮损伤标志物相关分析

目的　探讨2型糖尿病(DM)患者尿白蛋白/肌酐(Alb/Cr)排泄率与血管内皮损伤标志物的相关性。 方法　分别测定47例2型DM患者血浆P 选择素、vonWillebrand因子(vWF)、血清C反应蛋白(CRP)、Alb/Cr, 并根据尿Alb/Cr排泄率分为3组进行比较分析。结果　2型DM患者血浆P 选择素、vWF、血清CRP水平高于 正常对照组(P<0.05、0.002、0.001),并且与其尿Alb/Cr排泄率呈显著正相关。结论　血浆P 选择素、vWF和 血清CRP可作为观察2型DM并发血管病变以及严重程度的指标。     

RT-RAA联合CRISPR/Cas12a快速检测新型冠状病毒方法的建立与评价

摘要:目的建立一种基于反转录酶一重组酶介导等温核酸扩增技术( RT-RAA)联合簇状规则间隔的短回文重复序列(CRISPR)/Cas12a系统的快速检测新型冠状病毒(SARS-CoV-2)的方法,并对其进行评价。方法根据NCBI数据库中SARS-CoV-2的核衣壳(N)基因设计RT-RAA 引物和CRISPR来源RNA( crRNA),优化检测体系中乙酸镁( MgAc)用量、RT-RAA反应温度和时间以及LbCas12a反应温度。以10°~ 10 copies/μL重组质粒和其他呼吸道病原体分别评估该方法灵敏度和特异性。采用RT-RAA-CRISPR/Cas1 2a法和RT-PCR法对70份临床标本进行平行检测,比较两种方法的符合率。结果优化后的RT-RAA-CRISPR/Cas12a检测方法可在37 C条件下50 min内完成SARS-CoV-2检测。荧光法检测灵敏度为10 copies/μL,侧流层析法为1x 102 copies/μL。该方法能特异检测SARS-CoV-2, 与其他呼吸道病原体无交叉反应。RT-RAA-CRISPR/Cas12a法检测70份临床标本的结果与RT-PCR 法完全一致,符合率为100%。结论建立的RT-RAA-CRISPR/Cas 12a法检测SARS-CoV-2快速、经济、灵敏度高、特异性强,无需昂贵仪器设备,可由经验不足的人员操作,为临床快速诊断和现场大规模筛查SARS-CoV-2提供了新的思路和方法。     

Evaluation of Brugia malayi sheath protein (Shp-1) as a diagnostic antigen for human lymphatic filariasis

Lymphatic filariasis is the second leading cause of permanent long-term disability globally and control of this disease needs efficient diagnostic methods. In this study, abundantly expressing microfilarial sheath protein (Shp-1) from Brugia malayi was characterized as a filarial diagnostic candidate using samples from different clinical population. Monoclonal antibodies were developed against E. coli expressed recombinant Shp-1 in order to assess its efficiency in filarial antigen detection assay system. Endemic Normal (EN, n = 170), asymptomatic microfilaeremics (MF, n = 65), symptomatic chronic pathology (CP, n = 45) and non endemic normal (NEN, n = 10) sera were analyzed by antigen capture enzyme-linked immunosorbent assay. Of the 290 individuals, all MF individuals (both brugian and bancroftian) were positive in this assay followed by CP and EN. When compared with SXP-1 and Og4C3 antigen assays, all assays detected Wb MF correctly, Bm MF was detected by Shp-1 and SXP-1 assays, and only Shp-1 was able to detect EN (12%) and CP (29%). Results showed that this assay may be useful for monitoring prior to mass drug administration.     

Evaluation of a simultaneous detection kit for the glutamate dehydrogenase antigen and toxin A/B in feces for diagnosis of <Emphasis Type=Italic>Clostridium difficile</Emphasis> infection

Rapid detection kits for toxin A/B in feces are widely used as a diagnostic tool for Clostridium difficile infection (CDI). Their low sensitivity, however, has been considered a problem. In this study, we evaluated a new rapid diagnostic kit for simultaneous detection of the glutamate dehydrogenase (GDH) antigen and toxin A/B, C. DIFF QUIK CHEK COMPLETE. A total of 60 stool specimens from 60 patients with antibiotic-associated diarrhea were examined. Using C. difficile culture as the reference method, the GDH portion of this kit indicated a sensitivity, specificity, and negative predictive value of 100, 93.3, and 100%, respectively. The toxin A/B portion showed a sensitivity and specificity of 78.6 and 96.9%, respectively, compared to the culture results of toxin B-positive C. difficile (toxigenic culture). Of the 23 specimens that showed “dual positives” for GDH and toxin A/B, 22 were toxigenic culture positive, whereas C. difficile culture was negative in all the 28 specimens that showed “dual negatives” for GDH and toxin A/B. Of the nine “GDH-positive and toxin A/B-negative” specimens, six exhibited positive results by toxigenic culture. Results showing “dual positives” and “dual negatives” for GDH and toxin A/B can be reported as “true positive” and “true negative,” respectively, whereas additional testing for confirmation, such as toxigenic culture, is required for specimens with discrepant results. Diagnostic algorithms, utilizing the simultaneous detection kit for GDH and toxin A/B as an initial screening test, may be useful for accurate and efficient diagnosis of CDI as well as the control of healthcare-associated infections.