二代测序技术应用于脑脊液检测在结核性脑膜炎中的早期诊断价值

目的评价二代测序技术应用于脑脊液检测在结核性脑膜炎（TBM）患者中的早期诊断价值。 方法前瞻性纳入2018年2月2日至2018年8月2日于山东省胸科医院就诊的临床怀疑TBM的患者共50例,并跟踪随访其诊疗结局。送检脑脊液标本均进行二代测序,测序所得原始序列与病原微生物数据库进行对比得到最终结果。二代测序结果以检测到结核分枝杆菌复合群唯一比对序列为阳性,未检测到唯一比对序列为阴性。以符合脑脊液结核分枝杆菌培养阳性、涂片阳性、Xpert MTB/RIF检测阳性及结核分枝杆菌核酸检测阳性等4项中至少1项即为确诊TBM患者;临床可疑TBM且抗结核治疗有效为临床诊断患者;有其他病原学依据或临床排除TBM者为非TBM患者。分析二代测序在TBM早期诊断中的敏感性和特异度。 结果确诊为TBM患者22例中Xpert MTB/RIF检测阳性13例,培养阳性6例,结核分枝杆菌核酸PCR检测阳性5例,临床诊断为TBM患者12例,非TBM患者16例。在确诊及临床诊断患者中,二代测序技术检测到结核分枝杆菌复合群系列20例,敏感性为58.8%（20/34）,特异度为100%（16/16）。在确诊患者中,二代测序的敏感性为63.6%（14/22）;在同步进行结核分枝杆菌培养、Xpert MTB/RIF检测与二代测序的50例标本中,以临床诊断为标准,3种方法的特异度均为100%（16/16）;传统方法、Xpert MTB/RIF检测及二代测序的敏感性分别为29.4%（10/34）、38.2（13/24）和58.8（20/34）,前两种检测方法与二代测序敏感性差异均有统计学意义（McNemar检验：χ² = 8.333、P = 0.013,χ² = 8.333、P = 0.065）。传统方法与二代测序联合检测的敏感性高达82.4%（28/34）。 结论二代测序技术能够较快速地检测脑脊液中的结核分枝杆菌复合群,且其敏感性和特异度均较高,可作为TBM的早期诊断指标。二代测序联合传统检测方法可提高检出率。     

宏基因组二代测序技术对骨关节感染诊断的潜在应用价值

目的:评价宏基因组二代测序(mNGS)在骨关节感染诊断中的潜在应用价值。方法:回顾性分析青岛市胸科医院脊柱外科2019年1月至12月临床确诊为骨关节感染的37例住院患者临床资料。所有患者均经过穿刺或手术途径获取感染病灶核心部位组织样本,分别对样本进行分枝杆菌、需氧菌和厌氧菌常规培养,结核分枝杆菌(MTB)-DNA扩增检...     

高通量二代测序技术在泌尿生殖道无乳链球菌筛查中的应用

目的探讨高通量二代测序技术(NGS)在泌尿生殖道无乳链球菌(GBS)筛查中的应用价值。方法运用高通量二代测序技术检测58份男性尿道分泌物、30份处女和20份孕妇阴道分泌物中的微生物,同时进行GBS荧光定量聚合酶链反应(FQ-PCR)和传统细菌培养鉴定。结果GBS在NGS中的检出率为18.5%(20/108),在FQ-PCR中的检出率为20.4%(22/108),在传统细菌培养鉴定中检出率为4.6%(5/108)。NGS与FQ-PCR两种分子生物学方法在男性尿道分泌物、处女和孕妇阴道分泌物GBS筛查鉴定中符合率为100%、30%和0%。男性尿道分泌物中的菌群数量最多,孕妇阴道分泌物中的菌群数量最少。男女泌尿生殖道均有瑞士乳酸杆菌、惰性乳酸杆菌、copri普雷沃菌、无乳链球菌、婴儿链球菌等相同菌群存在。结论高通量二代测序技术具有高通量、高准确度和高灵敏度的特点,适合微生物菌群及物种的筛选,男、女泌尿生殖道均有无乳链球菌定植,且菌群结构部分相同。     

二代测序技术在诊断假体周围感染中的应用

[目的]探讨二代测序技术在关节置换术后假体周围感染诊断中的应用价值。[方法]选取2018年1月～2018年12月在本院就诊的人工关节置换术后关节疼痛、红肿、怀疑假体周围感染的患者。依据美国肌肉、骨骼感染病学会(MSIS)诊断标准,最终共计22例患者确诊为人工关节置换术后假体周围感染。其中男10例,女12例;膝关节感染16例,髋关节感染6例。所有22例PJI患者均行关节液二代测序和细菌培养。分别记录二代测序及细菌培养结果,以检测或培养出微生物为阳性,未检测或培养出微生物为阴性,记录阳性例数。分别计算二代测序技术和细菌培养在PJI患者中的阳性检出率以及在髋膝不同部位的阳性检出率,并通过配对卡方检验进行统计学分析。[结果]总计22例PJI患者中,18例患者二代测序检出微生物,二代测序在PJI患者中的阳性检出率为18/22(81.82%);关节液细菌培养阳性9例,关节液细菌培养在PJI患者中的阳性检出率为9/22(40.91%)。配对卡方检验提示两种方法在PJI患者中的检出率差异有统计学意义(P=0.022)。[结论]二代测序技术在PJI患者的诊断中具有更高的细菌检出率,在PJI患者病原微生物学检查中有更大的诊断价值。     

非小细胞肺癌细胞A549顺铂耐药潜在机制的全转录组测序分析

目的 通过全转录组测序分析比较野生型A549细胞和顺铂耐药A549细胞(A549/DPP)表达谱的差别,揭示非小细胞肺癌(NSCLC)顺铂耐药潜在机制.方法 首先建立A549/DDP细胞系,对A549和A549/DDP进行全转录组测序,分别对lncRNA-seq,circRNA-seq和miRNA-seq数据进行差异表...     

循环肿瘤DNA监测转移性乳腺癌治疗过程中肿瘤负荷量动态变化的探索性研究

目的探讨转移性乳腺癌患者在接受治疗过程中利用高通量测序技术进行循环肿瘤DNA（ctDNA）检测的价值。 方法通过目标区域捕获结合第二代高通量测序技术,对转移性乳腺癌患者在进行治疗期间不同治疗节点的ctDNA进行测序,并与临床影像学指标进行对比。 结果本研究共纳入5例转移性乳腺癌患者不同时间点的13份血液样本。利用二代高通量测序技术成功在12份（92.3%）样本中检测出ctDNA,联合生物信息学分析出ctDNA在治疗过程中的各个突变克隆的变化趋势。 结论二代测序技术对检测转移性乳腺癌的突变谱有应用价值,ctDNA的动态变化能有效反映转移性乳腺癌患者在治疗过程中不同时期的肿瘤负荷量变化。     

非标记探针高分辨率熔解曲线技术检测HBV P区rt204位点耐药突变

目的 建立一种基于非标记探针高分辨率熔解曲线技术(HRM)检测HBV P区rt204位点耐药突变的方法.方法 构建野生株rt204M、突变株rt204I和rt204V的质粒,设计、优化探针.利用质粒作为标准品建立HRM特征性解链图谱.收集2010年5月至2012年5月宁波大学附属医院诊治的185例慢性乙型肝炎患者外周血样本共185份,用HRM技术检测所有样本,并与特征性解链图谱比对,筛选出rt204位点的突变,再经基因测序验证.采用配对x2检验比较两种方法的变异检出率.结果 突变株rt204I、rt204V和野生株rt204M的熔解温度(Tm)分别为58.0℃、60.6℃和62.5℃;185份样本中,HRM技术成功分析168份(90.8％),基因测序成功分析155份(83.8％),二者差异有统计学意义(P＜0.01);同时被两种方法检出的155份临床样本中,非标记探针HRM技术检测到野生株75份(rt204M),变异株80份(55份rt204I、25份rt204V),变异检出率为51.6％(80/155);基因测序法检测到野生株110份(rt204M),变异株45份(30份rt204I、15份rt204V),变异检出率为29.0％ (45/155),两种方法差异有统计学意义(P＜0.01).结论 非标记探针HRM技术是一种简单、灵敏、快速、特异的HBV P区rt204位点耐药突变检测方法.     

宏基因组二代测序技术联合椎间孔镜下扩创治疗腰椎原发性感染

目的 观察宏基因组二代测序技术联合椎间孔镜下扩创治疗腰椎原发性感染的临床疗效。方法 纳入自2019-01—2020-12应用宏基因组二代测序技术联合椎间孔镜下扩创治疗的24例考虑原发性腰椎感染。术中取出炎症病变组织,分别送细菌培养、抗酸染色、X-pert、宏基因组二代测序技术(Metagenomic next-generation sequencingm,mNGS)、病理检查,术后根据致病菌检测结果选用抗生素。将致病菌的检测分为mNGS组和常规组,常规组包含目前常用的检测方法之和,即血培养、组织细菌培养、布鲁氏杆菌试管凝集试验、T-spot、组织病理TB-DNA阳性、X-pert等,mNGS组采用宏基因组二代测序技术。结果 24例中有1例证实为脊柱夏科氏病并予以排除,其余23例均为原发性腰椎感染。术前血培养阳性3例(3/23,13.04%),布鲁氏杆菌试管凝集试验阳性3例(3/23,13.04%),T-spot阳性1例(1/23,4.35%)。术中标本,X-pert阳性1例(1/23,4.35%),组织培养阳性4例(4/23,17.39%),组织病理TB-DNA阳性3例(3/23,13...     

两个遗传性并多指(趾)家系致病基因突变分析

目的探讨2个并多指(趾)畸形(SPD)家系的致病基因。方法收集2019年1月、2020年12月就诊于临沂市人民医院的2个SPD家系的临床资料, 采集先证者及家系成员静脉血样本, 提取基因组DNA, 对先证者行全外显子组测序筛选候选基因变异;采用Sanger测序对2个家系成员验证其突变位点;采用生物信息学软件PolyPhen-2和PROVEAN对突变位点的致病性进行预测分析, 结合美国医学遗传学与基因组学学会(ACMG)指南对突变位点进行致病性判断。结果家系1三代成员中共有5例患者(男2例、女3例), 先证者为8岁女性, 表现为右手第3、4指并指, 指蹼融合和远端指甲融合, 其余手指活动自如, 双脚未见异常;家系2三代成员中共有4例患者(均为女性), 先证者为4岁女性, 表现为双手第3、4指并指, 示指侧弯。全外显子组测序分别在2个SPD家系中检出同源盒D13(HOXD13)基因c.917G>A和c.917G>T突变, 且2个突变均呈现基因型-表型共分离, 其中HOXD13基因c.917G>T突变未见数据库收录, 为新发杂合错义突变。生物信息学软件预测这2个突变位点均为...     

MALDI-TOF-MS技术对HBV耐药基因突变检测的价值

目的:探讨MALDI-TOF-MS技术对乙型肝炎病毒(HBV)耐药基因突变检测的价值.方法:依据HBV、HCV基因组多态性及基因突变现有研究结果,选择靶区域,筛选突变位点,MassARRAY Assay Design软件设计iPLEX引物,按iPLEX反应的操作说明进行PCR扩增、SAP反应、引物延伸、脱盐、点样,MALDI-TOF-MS采集、分析iPLEX反应物的数据,判读基因变异位点.收集本院抗病毒药物单药或多药耐药患者血清30份,将MALDI-TOF-MS技术判读基因变异位点进行DNA测序,将结果与MALDI-TOF-MS检测结果进行对比.结果:利用MALDI-TOF-MS技术一次性可检测30份血清样本的HBV基因突变,在30份样本中,5份检测结果不一致,其中2份是MALDI-TOF-MS技术未检测到的基因突变点,1份是2个检测位点不一致.结论:MALDI-TOF-MS技术具有灵敏度高、准确度高及分辨率高等特点,为生命科学等领域提供了一种强有力的分析测试手段.     

Past and future impact of next‐generation sequencing in head and neck cancer

Progress in sequencing technology is intrinsically linked to progress in understanding cancer genomics. The purpose of this review was to discuss the development from Sanger sequencing to next‐generation sequencing (NGS) technology. We highlight the technical considerations for understanding reports using NGS. We discuss the findings of studies in head and neck cancer using NGS as well as The Cancer Genome Atlas. Finally we discuss future routes for research utilizing this methodology and the potential impact of this. © 2015 Wiley Periodicals, Inc. Head Neck 38 : E2395–E2402, 2016     

How Far Should We Explore Hypospadias? Next-generation Sequencing Applied to a Large Cohort of Hypospadiac Patients

BackgroundNext-generation sequencing (NGS) is generally used for patients with severe disorders of sex development (DSD). However, NGS has not been applied extensively for patients with hypospadias only, and most affected children do not benefit from an etiological diagnosis.ObjectiveTo evaluate the clinical usefulness of NGS for patients with hypospadias, regardless of severity.Design, setting, and participantsProspective multicenter research included 293 children with glandular to penoscrotal hypospadias (no undescended testis and no micropenis). After excluding likely pathogenic androgen receptor (AR) variants by Sanger sequencing, an NGS panel tested 336 genes including unexplored candidates in 284 patients.Outcome measurements and statistical analysisThe rate of pathogenic and likely pathogenic variants was assessed using REVEL, ClinVar, and in-house tools (Captain-ACHAB, MobiCNV, and MobiDetails).Results and limitationsLikely pathogenic variants were identified in 16 (5.5%) patients with both Sanger sequencing and NGS taken into account. Some genes were related to DSD (AR, NR5A1, HSD17B3, and MAMLD1), but reverse phenotyping revealed two syndromic disorders with midline defects (MID1) and alteration in the retinoic acid signaling pathway (RARA). Coverage analysis revealed an 18q deletion. Identification of likely pathogenic variants increased with hypospadias severity. Other variants of unknown significance (VUSs) in genes implicated in hypogonadotropic hypogonadism, Noonan syndrome, and genital tubercle development were also identified. Genetic study mainly focused on exonic variants, and most cases remain unexplained.ConclusionsNGS reveals minor forms of DSD, undiagnosed syndromes, or candidate rare variants in new genes, indicating that even patients with mild hypospadias benefit from advanced sequencing techniques. Early molecular diagnosis would help improve follow-up at puberty and medical counseling for initially undiagnosed syndromes. Future studies will improve the diagnosis by investigating the contribution of VUSs.Patient summaryNext-generation sequencing enables simultaneous testing of numerous genes and should not be limited to disorders of sex development cases. Even patients with mild hypospadias would benefit from early diagnosis of a genetic defect implicated in sex development or other syndromes.     

基因二代测序技术在假体周围感染诊断中的价值

目的通过与细菌培养及血清生物学标志对比,探讨二代测序技术(NGS)对人工关节置换术后假体周围感染(PJI)的诊断价值。 方法选取2017年7月到2019年12月在聊城市人民医院关节外科因假体周围感染或无菌性松动行关节翻修手术,排除初始关节液无法采集到,通过关节内注射生理盐水获得样本的患者及其他部位存在感染病灶的患者,共纳入患者35人(35例)根据美国肌骨骼感染协会(MSIS)的诊断标准,15例患者纳入感染组,20例患者纳入非感染组。术前两组患者常规检查血沉(ESR)、C反应蛋白(CRP)、降钙素原(PCT)、白介素6(IL-6)及D-二聚体(D-Dimer)。所有患者术前均行关节穿刺,穿刺液检测白细胞计数、白细胞分类、细菌培养及NGS。计算ESR、CRP、PCT、IL-6及D-Dimer的受试者操作特性曲线(ROC)的曲线下面积(AUC)。计算NGS、细菌培养及各项血清学标志物的诊断精确度、敏感性及特异性。 结果髋关节19例(54.3%),膝关节16例(45.7%)。男性21例(60.0%),女性14例(40.0%),年龄67.0(62.0,74.0)岁。感染组15例患者中NGS结果阳性14例(93.3%),细菌培养结果阳性7例(46.7%)。非感染组NGS结果阴18例(90.0%)。ESR及D二聚体的AUC分别为0.667和0.572(均为P>0.05)。CRP、IL-6及PCT的AUC分别为0.827、0.767及0.808(均为P<0.01)。NGS、细菌培养、CRP、IL-6及PCT的精确度分别为0.91、0.74、0.77、0.74及0.83。NGS与CRP、IL-6、PCT、细菌培养两两比较,总体检测结果差异有统计学意义(P<0.01)。NGS与CRP、IL-6、PCT、细菌培养两两比较,NGS敏感性更高(P<0.05)。NGS与CRP比较特异性更好(P<0.05)。NGS与IL-6、PCT及细菌培养比较,特异性差异无统计学意义(P>0.05)。 结论NGS比细菌培养及常用的血清学标志物有更高的精确度及敏感性,在PJI的诊断中具有更大的价值。     

The diagnostic value of blood sample NGS in patients with early periprosthetic joint infection after total hip arthroplasty

The diagnostic value of next-generation sequencing (NGS) of blood samples from patients with periprosthetic joint infection (PJI) after total hip arthroplasty (THA) was evaluated by comparing it with drainage fluid NGS and bacterial culture. The study was designed as a retrospective diagnostic test. Thirty-six infected patients were diagnosed with PJI according to the Musculoskeletal Infection Society (MSIS) criteria and 57 volunteers were included in our study. NGS and bacterial culture were chosen to detect PJI after THA. Blood samples and drainage fluid were collected for NGS, and the drainage fluid, which was collected at the same time as the NGS drainage fluid sample, was used for bacterial culture. The primary outcomes of interest were sensitivity, specificity, and accuracy. In the infection group, 31 patients showed positive results by blood sample NGS, 33 patients showed positive results by drainage fluid NGS, and 17 patients showed positive bacterial culture results. In the control group, the results of 2 blood sample NGS, 16 drainage fluid NGS, and 3 bacterial cultures were positive. The sensitivity, specificity, and accuracy of the blood sample were 0.86, 0.96, and 0.92, respectively. The sensitivity, specificity, and accuracy of the drainage fluid samples were 0.92, 0.72, and 0.80, respectively. The sensitivity, specificity, and accuracy of bacterial culture were 0.47, 0.95, and 0.79, respectively. The study demonstrated that both the sensitivity and specificity of NGS were higher than those of bacterial culture, regardless of the kind of sample. Compared with drainage fluid NGS, the sensitivity of blood sample NGS was slightly lower (0.86 vs 0.92), but blood sample NGS showed higher specificity (0.96 vs 0.72). In total, the diagnostic value of blood sample NGS was superior to that of drainage fluid NGS and bacterial culture. The majority of infected patients could be identified by blood sample NGS. Moreover, because of its high specificity, blood sample NGS can not only detect infectious bacteria but also distinguish infectious from non-infectious bacteria, which is dramatically different from using drainage fluid NGS.     

Diagnostic Value of Next‐Generation Sequencing in Periprosthetic Joint Infection: A Systematic Review

Next‐generation sequencing (NGS) has developed rapidly in the last decade and is emerging as a promising diagnostic tool for periprosthetic joint infection (PJI). However, its diagnostic value for PJI is still uncertain. This systematic review aimed to explore the diagnostic value of NGS for PJI and verify its accuracy for culture‐negative PJI patients. We conducted this systematic review in accordance with the Preferred Reporting Items for Systematic Reviews and Meta‐Analysis (PRISMA) guidelines. Medline, Embase, and Cochrane Library were searched to identify diagnostic technique studies evaluating the accuracy of NGS in the diagnosis of PJI. The diagnostic sensitivity, specificity, and positive and negative predictive values were estimated for each article. The detection rate of NGS for culture‐negative PJI patients or PJI patients with antibiotic administration history was also calculated. Of the 87 identified citations, nine studies met the inclusion criteria. The diagnostic sensitivities and specificities of NGS ranged from 63% to 96% and 73% to 100%, respectively. The positive and negative predictive values ranged from 71% to 100% and 74% to 95%, respectively. The detection rate of NGS for culture‐negative PJI patients in six studies was higher than 50% (range from 82% to 100%), while in three studies it was lower than 50% (range from 9% to 31%). Also, the detection rate of NGS for PJIs with antibiotic administration history ranged from 74.05% to 92.31%. In conclusion, this systematic review suggests that NGS may have the potential to be a new tool for the diagnosis of PJI and should be considered to be added to the portfolio of diagnostic procedures. Furthermore, NGS showed a favorable diagnostic accuracy for culture‐negative PJI patients or PJI patients with antibiotic administration history. However, due to the small sample sizes of studies and substantial heterogeneity among the included studies, more research is needed to confirm or disprove these findings.     

Molecular Detection of Tick‐Borne Pathogens of the Family Anaplasmataceae in Brazilian Brown Brocket Deer (Mazama gouazoubira,Fischer, 1814) and Marsh Deer (Blastocerus dichotomus,Illiger, 1815)

Deer are important natural reservoir hosts of Anaplasmataceae. The present study used nested PCR and nucleotide sequencing to evaluate the occurrence of Anaplasmataceae species in 23 free‐living and six captive specimens of the cervids Mazama gouazoubira and Blastocerus dichotomus in Minas Gerais State, Brazil. Blood samples were tested for the presence of Ehrlichia and Anaplasma spp. using nPCR assays and sequencing of the msp4, msp1 and 16S rRNA genes. The identity of each sequence was confirmed by comparison with sequences available from GenBank using BLAST software. Of the animals investigated, 93.1% (27/29) were infected with haemoparasites including Anaplasma marginale (79.3%), Ehrlichia chaffeensis (3.4%), Anaplasma bovis (3.4%) and Anaplasma spp. (assigned to A. platys and A. phagocytophilum) (17.2%). Co‐infection occurred in 20% (6/29) of the deer examined. Four (13.8%) were infected with A. marginale and Anaplasma sp., one (3.4%) was infected with A. marginale and E. chaffeensis, and one (3.4%) was infected with A. marginale and A. bovis. The results of the present study suggest that cross‐protection does not occur in these deer. Immunological cross‐reaction occurs when sera are tested diagnostically because these bacteria are closely related taxonomically, reinforcing the importance of molecular diagnosis followed by nucleotide sequencing.     

Hybrid minigene splicing assay verifies the pathogenicity of a novel splice site variant in the COL1A1 gene of a chinese patient with osteogenesis imperfecta type I

BackgroundOsteogenesis imperfecta (OI) is a rare genetic bone disease associated with brittle bones and fractures. Among all known types, OI type I is the most common type and characterized by increased bone fragility, low bone mass, distinctly blue-gray sclera, and susceptibility to conductive hearing loss beginning in adolescence. Mutations in genes encoding type I collagen (COL1A1 and COL1A2) contribute to the main pathogenic mechanism of OI.MethodsSubtle mutation of the COL1A1 gene in the proband was detected by targeted next-generation sequencing (NGS) and confirmed by Sanger sequencing. We then assessed the effect of the mutation on the splicing of the COL1A1 gene by bioinformatics prediction and hybrid minigene splicing assay (HMSA).ResultsA novel splice site mutation c.1821+1 G>C was discovered in the proband by NGS and further confirmed by Sanger sequencing, which was also simultaneously identified from the proband's mother and elder sister. Bioinformatics predicted that this mutation would result in a disappearance of the 5′ donor splice site in intron 26, thereby leading to abnormal splicing and generation of premature stop codon. The follow-up experimental data generated by HMSA was consistent with this prediction.ConclusionOur study identified a novel splice site mutation that caused OI type I in the proband by abnormal splicing and demonstrated that combined applications of NGS, bioinformatics and HMSA are comprehensive and effective methods for diagnosis and aberrant splicing study of OI.