qPCR快速检测金黄色葡萄球菌及MRSA方法的建立及评价

目的建立q PCR法快速检测金黄色葡萄球菌及其mec A基因,以期快速精准诊断金黄色葡萄球菌感染及初步判断耐药情况。方法从NCBI数据库下载金黄色葡萄球菌nuc、atl、ica B、fnb A、hla、srap基因序列用于鉴定标志筛选,mec A基因序列用于MRSA标志筛选;经DNA MAN比对后,选择各基因保守区分别设计1~2套引物、探针,建立单重及双重q PCR,用临床分离株及标准菌株筛选出检测性能最好的基因片段作为检测标志物,建立金黄色葡萄球菌鉴定和耐药双重q PCR检测方法,并进行性能评价。结果经筛选,金黄色葡萄球菌atl基因(CP009361.1:1010217~1010341)、mec A基因(KF058908.1:1715~1843)两片段检测性能最优,将其作为标志物建立双重q PCR法。该方法的检测下限均低至4 copies/反应;扩增线性范围均达2.0×102~8copies/m L。335份金黄色葡萄球菌(含94份MRSA)培养阳性的患者样本中,q PCR法分别检出SA 335份,MRSA 94份;95份金黄色葡萄球菌培养阴性的患者样本中,q PCR法分别检出SA 17份,MRSA 4份,经PCR产物测序,与标准菌株同源性均≥90%。双重q PCR法从样品处理到报告结果≤2.0 h。结论 q PCR法方法简便、快速、灵敏度高、特异性好,可望提高金黄色葡萄球菌感染的诊断能力并实现快速检测,为尽早精准治疗赢得时间。     

Mechanisms and biomarkers of immune quiescence in kidney transplantation

This review discusses the current understanding of biomarkers of immune quiescence based on reviews of published literature in kidney transplant operational tolerance and mechanistic studies based on a better characterization of the stable, well-functioning renal allograft.     

Comparison of a healthy miRNome with melanoma patient miRNomes: are microRNAs suitable serum biomarkers for cancer?

MiRNAs are increasingly recognized as biomarkers for the diagnosis of cancers where they are profiled from tumor tissue (intracellular miRNAs) or serum/plasma samples (extracellular miRNAs). To improve detection of reliable biomarkers from blood samples, we first compiled a healthy reference miRNome and established a well-controlled analysis pipeline allowing for standardized quantification of circulating miRNAs. Using whole miRNome and custom qPCR arrays, miRNA expression profiles were analyzed in 126 serum, whole blood and tissue samples of healthy volunteers and melanoma patients and in primary melanocyte and keratinocyte cell lines. We found characteristic signatures with excellent prognostic scores only in late stage but not in early stage melanoma patients. Upon comparison of melanoma tissue miRNomes with matching serum samples, several miRNAs were identified to be exclusively tissue-derived (miR-30b-5p, miR-374a-5p and others) while others had higher expression levels in serum (miR-3201 and miR-122-5p). Here we have compiled a healthy and widely applicable miRNome from serum samples and we provide strong evidence that levels of cell-free miRNAs only change significantly at later stages of melanoma progression, which has serious implications for miRNA biomarker studies in cancer.     

BRAF mutation testing with a rapid,fully integrated molecular diagnostics system

Fast and accurate diagnostic systems are needed for further implementation of precision therapy of BRAF-mutant and other cancers. The novel Idylla^TM BRAF Mutation Test has high sensitivity and shorter turnaround times compared to other methods. We used Idylla to detect BRAF V600 mutations in archived formalin-fixed paraffin-embedded (FFPE) tumor samples and compared these results with those obtained using the cobas 4800 BRAF V600 Mutation Test or MiSeq deep sequencing system and with those obtained by a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory employing polymerase chain reaction–based sequencing, mass spectrometric detection, or next-generation sequencing. In one set of 60 FFPE tumor samples (15 with BRAF mutations per Idylla), the Idylla and cobas results had an agreement of 97%. Idylla detected BRAF V600 mutations in two additional samples. The Idylla and MiSeq results had 100% concordance. In a separate set of 100 FFPE tumor samples (64 with BRAF mutation per Idylla), the Idylla and CLIA-certified laboratory results demonstrated an agreement of 96% even though the tests were not performed simultaneously and different FFPE blocks had to be used for 9 cases. The Idylla^TM BRAF Mutation Test produced results quickly (sample to results time was about 90 minutes with about 2 minutes of hands on time) and the closed nature of the cartridge eliminates the risk of PCR contamination. In conclusion, our observations demonstrate that the Idylla test is rapid and has high concordance with other routinely used but more complex BRAF mutation–detecting tests.