Diagnosis and treatment of refractory infectious diseases using nanopore sequencing technology:Three case reports

BACKGROUND Infectious diseases are still one of the greatest threats to human health,and the etiology of 20%of cases of clinical fever is unknown;therefore,rapid identification of pathogens is highly important.Traditional culture methods are only able to detect a limited number of pathogens and are time-consuming;serologic detection has window periods,false-positive and false-negative problems;and nucleic acid molecular detection methods can detect several known pathogens only once.Three-generation nanopore sequencing technology provides new options for identifying pathogens.CASE SUMMARY Case 1:The patient was admitted to the hospital with abdominal pain for three days and cessation of defecation for five days,accompanied by cough and sputum.Nanopore sequencing of the drainage fluid revealed the presence of orallike bacteria,leading to a clinical diagnosis of bronchopleural fistula.Cefoperazone sodium sulbactam treatment was effective.Case 2:The patient was admitted to the hospital with fever and headache,and CT revealed lung inflammation.Antibiotic treatment for Streptococcus pneumoniae,identified through nanopore sequencing of cerebrospinal fluid,was effective.Case 3:The patient was admitted to our hospital with intermittent fever and an enlarged neck mass that had persisted for more than six months.Despite antibacterial treatment,her symptoms worsened.The nanopore sequencing results indicate that voriconazole treatment is effective for Aspergillus brookii.The patient was diagnosed with mixed cell type classical Hodgkin's lymphoma with infection.CONCLUSION Three-generation nanopore sequencing technology allows for rapid and accurate detection of pathogens in human infectious diseases.

Detection of Novel BEST1 Variations in Autosomal Recessive Bestrophinopathy Using Third-generation Sequencing

Objective:Autosomal recessive bestrophinopathy(ARB),a retinal degenerative disease,is characterized by central visual loss,yellowish multifocal diffuse subretinal deposits,and a dramatic decrease in the light peak on electrooculogram.The potential pathogenic mechanism involves mutations in the BEST1 gene,which encodes Ca2+-activated Cl−channels in the retinal pigment epithelium(RPE),resulting in degeneration of RPE and photoreceptor.In this study,the complete clinical characteristics of two Chinese ARB families were summarized.Methods:Pacific Biosciences(PacBio)single-molecule real-time(SMRT)sequencing was performed on the probands to screen for disease-causing gene mutations,and Sanger sequencing was applied to validate variants in the patients and their family members.Results:Two novel mutations,c.202T>C(chr11:61722628,p.Y68H)and c.867+97G>A,in the BEST1 gene were identified in the two Chinese ARB families.The novel missense mutation BEST1 c.202T>C(p.Y68H)resulted in the substitution of tyrosine with histidine in the N-terminal region of transmembrane domain 2 of bestrophin-1.Another novel variant,BEST1 c.867+97G>A(chr11:61725867),located in intron 7,might be considered a regulatory variant that changes allele-specific binding affinity based on motifs of important transcriptional regulators.Conclusion:Our findings represent the first use of third-generation sequencing(TGS)to identify novel BEST1 mutations in patients with ARB,indicating that TGS can be a more accurate and efficient tool for identifying mutations in specific genes.The novel variants identified further broaden the mutation spectrum of BEST1 in the Chinese population.

Surveillance of emerging SARS-CoV-2 variants by nanopore technology-based genome sequencing

Objective:To surveill emerging variants by nanopore technology-based genome sequencing in different COVID-19 waves in Sri Lanka and to examine the association with the sample characteristics,and vaccination status.Methods:The study analyzed 207 RNA positive swab samples received to sequence laboratory during different waves.The N gene cut-off threshold of less than 30 was considered as the major inclusion criteria.Viral RNA was extracted,and elutes were subjected to nanopore sequencing.All the sequencing data were uploaded in the publicly accessible database,GISAID.Results:The Omicron,Delta and Alpha variants accounted for 58%,22%and 4%of the variants throughout the period.Less than 1%were Kappa variant and 16%of the study samples remained unassigned.Omicron variant was circulated among all age groups and in all the provinces.Ct value and variants assigned percentage was 100%in Ct values of 10-15 while only 45%assigned Ct value over 25.Conclusions:The present study examined the emergence,prevalence,and distribution of SARS-CoV-2 variants locally and has shown that nanopore technology-based genome sequencing enables whole genome sequencing in a low resource setting country.

Application of next-generation sequencing technology to precision medicine in cancer: joint consensus of the Tumor Biomarker Committee of the Chinese Society of Clinical Oncology

Next-generation sequencing(NGS) technology is capable of sequencing millions or billions of DNA molecules simultaneously.Therefore, it represents a promising tool for the analysis of molecular targets for the initial diagnosis of disease, monitoring of disease progression, and identifying the mechanism of drug resistance. On behalf of the Tumor Biomarker Committee of the Chinese Society of Clinical Oncology(CSCO) and the China Actionable Genome Consortium(CAGC), the present expert group hereby proposes advisory guidelines on clinical applications of NGS technology for the analysis of cancer driver genes for precision cancer therapy. This group comprises an assembly of laboratory cancer geneticists, clinical oncologists, bioinformaticians,pathologists, and other professionals. After multiple rounds of discussions and revisions, the expert group has reached a preliminary consensus on the need of NGS in clinical diagnosis, its regulation, and compliance standards in clinical sample collection. Moreover, it has prepared NGS criteria, the sequencing standard operation procedure(SOP), data analysis, report, and NGS platform certification and validation.

Innovation and development of the third-generation hybrid rice technology

The breeding and large-scale application of hybrid rice contribute significantly to the food supply worldwide.Currently,hybrid seed production uses cytoplasmic male sterile(CMS)lines or photoperiod/thermo-sensitive genic male sterile(PTGMS)lines as female parent.Despite huge successes,both systems have intrinsic problems.CMS systems are mainly restricted by the narrow restorer resources that make it difficult to breed superior hybrids,while PTGMS systems are limited by conditional sterility of the male sterile lines that makes the propagation of both PTGMS seeds and hybrid seeds vulnerable to unpredictable climate changes.Recessive nuclear male sterile(NMS)lines insensitive to environmental conditions are widely distributed and are ideal for hybrid rice breeding and production,but the lack of effective ways to propagate the pure NMS lines in a large scale renders it impossible to use them for hybrid rice production.The development of"the third-generation hybrid rice technology"enables efficient propagation of the pure NMS lines in commercial scale.This paper discusses the establishment of"the thirdgeneration hybrid rice technology"and further innovations.This new technology breaks the limitations of CMS and PTGMS systems and will bring a big leap forward in hybrid rice production.

Application of Nanopore Sequencing Technology in the Clinical Diagnosis of Infectious Diseases

Infectious diseases are an enormous public health burden and a growing threat to human health worldwide.Emerging or classic recurrent pathogens,or pathogens with resistant traits,challenge our ability to diagnose and control infectious diseases.Nanopore sequencing technology has the potential to enhance our ability to diagnose,interrogate,and track infectious diseases due to the unrestricted read length and system portability.This review focuses on the application of nanopore sequencing technology in the clinical diagnosis of infectious diseases and includes the following:(i)a brief introduction to nanopore sequencing technology and Oxford Nanopore Technologies(ONT)sequencing platforms;(ii)strategies for nanopore-based sequencing technologies;and(iii)applications of nanopore sequencing technology in monitoring emerging pathogenic microorganisms,molecular detection of clinically relevant drug-resistance genes,and characterization of disease-related microbial communities.Finally,we discuss the current challenges,potential opportunities,and future outlook for applying nanopore sequencing technology in the diagnosis of infectious diseases.

Advances in single-cell sequencing technology in microbiome research

With the rapid development of histological techniques and the widespread applica-tion of single-cell sequencing in eukaryotes,researchers desire to explore individual microbial.genotypes and functional expression,which deepens our understanding of microorganisms.In this review,the history of the development of microbial detection technologies was revealed and the difficulties in the application of single-cell sequencing in microorganisms were dissected as well.Moreover,the characteristics of the currently emerging microbial single-cell sequencing(Microbe-seq)technology were summarized,and the prospects of the application of Microbe-seq in microorganisms were distilled based on the current development status.Despite its mature development,the Microbe-seq technology was still in the optimization stage.A retrospective study was conducted,aiming to promote the widespread application of single-cell sequencing in microorganisms and facilitate further improvement in the technol-ogy.

Thoughts on the Development of Bridge Technology in China

In the history of bridge engineering, demand has always been the primary driving force for development. Driven by the huge demand for construction since China’s reform and opening-up, Chinese bridge has leapt forward both quantitatively and qualitatively in three major stages, by completing the transition from “follower” to “competitor,” and nally to “leader.” A new future is emerging for Chinese bridge engi- neering. As an important part of China’s transportation infrastructure, the bridge engineering industry is facing challenges in this new era on how to support the construction of a new form of transportation. This paper provides a summary of the status of bridge technology in China, based on a basic analysis of stock demand, incremental demand, and management demand. It is our belief that the Chinese bridge engi- neering industry must ful ll three outstanding requirements: construction ef ciency, management effec- tiveness, and long-term service. Intelligent technology based on information technology provides a new opportunity for innovation in bridge engineering. As a result, the development path of bridge engineering needs to be changed. This paper puts forward the idea of developing a third-generation bridge project that is characterized by intelligence, and discusses this project’s implications, development focus, and plan. In this way, this work provides a direction for the improvement of the core competitiveness of China’s bridge engineering industry.

Current status and future perspectives for sequencing livestock genomes

Only in recent years, the draft sequences for several agricultural animals have been assembled. Assembling an individual animal＇s entire genome sequence or specific region（s） of interest is increasingly important for agricultura researchers to perform genetic comparisons between animals with different performance. We review the current status for several sequenced agricultural species and suggest that next generation sequencing （NGS） technology with decreased sequencing cost and increased speed of sequencing can benefit agricultural researchers. By taking advantage of advanced NGS technologies, genes and chromosomal regions that are more labile to the influence of environmental factors could be pinpointed. A more long term goal would be addressing the question of how animals respond at the molecular and cellular levels to different environmental models （e.g. nutrition）. Upon revealing important genes and gene-environment interactions, the rate of genetic improvement can also be accelerated. It is clear that NGS technologies will be able to assist animal scientists to efficiently raise animals and to better prevent infectious diseases so that overall costs of animal production can be decreased.

Application of Biological Nanopore Sequencing Technology in the Detection of Microorganisms

Environmental pollution and the spread of pathogenic microorganisms pose a significant threat to the health of humans and the planet.Thus,understanding and detecting microorganisms is crucial for maintaining a healthy living environment.Nanopore sequencing is a single-molecule detection method developed in the 1990s that has revolutionized various research fields.It offers several advantages over traditional sequencing methods,including low cost,label-free,time-saving detection speed,long sequencing reading,real-time monitoring,convenient carrying,and other significant advantages.In this review,we summarize the technical principles and characteristics of nanopore sequencing and discuss its applications in amplicon sequencing,metagenome sequencing,and whole-genome sequencing of environmental microorganisms,as well as its in situ application under some special circumstances.We also analyze the advantages and challenges of nanopore sequencing in microbiology research.Overall,nanopore sequencing has the potential to greatly enhance the detection and understanding of microorganisms in environmental research,but further developments are needed to overcome the current challenges.

A KIND OF PAPR REDUCTION METHOD BASED ON PRUNING WPM AND PTS TECHNOLOGY

Wavelet packet multicarrier system gains widespread concern because of its better resistance performance to Inter-Symbol Interference (ISI) and Inter-Carrier Interference (ICI), as well as the higher spectrum efficiency. However, multicarrier system has a high Peak to Average Power Ratio (PAPR), which will lead to many problems such as lower system performance. In order to solve the problem, a kind of PAPR reduction method based on pruning Wavelet Packet Modulation (WPM) and Partial Transmit Sequences (PTS) technology is proposed in this paper, through proper pruning of the full-tree structure of wavelet packet modulation in the PTS technology to reduce the number of nodes in the system, and finally improve the reduction effect of PAPR. Simulation results show that when Complementary Cumulative Distribution Function (CCDF) is 10 -3 , PTS based on pruning WPM compared with PTS technique and pruning technique has improved about 1 dB and 1.5 dB, which will not affect the system's Bit Error Rate (BER) performance in the wavelet packet multicarrier system.

设施西瓜连作土壤生化性质及微生物群落变化

为探索设施连作西瓜土壤微生态环境变化,通过高通量测序对连作组(连作10年设施西瓜土壤)和对照组(连作10年设施西瓜棚外相同土质的露天西瓜土壤)样品进行测序分析,比较2组土壤细菌和真菌的群落结构差异,测定土壤理化性质及酶活性。结果表明,连作组土壤pH、铵态氮、速效钾、微生物量碳、土壤酶活性极显著低于对照,而硝态氮和有效磷极显著高于对照组。连作组土壤中细菌ACE指数和Chaol指数均极显著高于对照组,土壤中真菌ACE指数、Chaol指数、Simpson指数和Shannon指数均低于对照组。土壤微生物优势物种组成改变,连作组土壤细菌变形菌门、芽单胞菌门、绿弯菌门及硝化螺旋菌门相对丰度较对照组分别增加了9.80%、20.83%、21.76%和27.12%(P<0.05),而酸杆菌门和疣微菌门较对照组分别减少了21.7%和26.27%(P<0.05);连作组土壤真菌子囊菌门相对丰度较对照组增加了10.96%(P<0.05),担子菌门和罗兹菌门的相对丰度分别较对照组减少了13.42%和93.77%(P<0.05)。相关性分析显示,优势菌门除Latescibacteria和己科河菌门丰度与所有土壤环境因子相关性均不显著外,其他优势菌群都与所测土壤环境因子(有机质除外)存在密切相关关系,土壤pH、铵态氮、有效磷、速效钾是影响土壤中微生物群落的主要因素,担子菌门和绿弯菌门能够指示土壤微生物量,微生物群落结构的变化对土壤酶活性有较大的影响。综上,设施西瓜连作10年土壤理化性质、酶活性及微生物群落结构皆显著变化,西瓜连作障碍是多种因素综合作用的结果。

磁共振T1rho序列、T2 mapping技术在膝关节早期软骨退变早期诊断中的评估价值

目的探讨磁共振T1rho序列、T2 mapping技术在膝关节早期软骨退变早期诊断中的评估价值。方法选取2020年1月至2022年11月在我院治疗的110例膝关节早期软骨退变患者(观察组),按照疾病严重程度分为轻度退变组及重度退变组,另选取同期在我院进行体检的64例健康者为对照组,受试者均行磁共振T1rho序列、T2 mapping技术扫描,测量受试者T2值及T1ρ值,探讨磁共振T1rho序列、T2 mapping技术在膝关节早期软骨退变早期诊断中的评估价值。结果观察组股骨外侧面、股骨内侧面、胫骨外侧面、胫骨内侧面、髋骨面T2值均明显高于对照组(P<0.05),重度退变亚组患者股骨外侧面、股骨内侧面、胫骨外侧面、胫骨内侧面、髋骨面T2值显著高于轻度退变者(P<0.05);观察组股骨内踝负重区、股骨内踝非负重区、股骨外踝负重区、股骨外踝非负重区、胫骨外侧平台区、胫骨内侧平台区、髌后软骨区磁共振T1ρ值明显高于对照组(P<0.05),重度退变亚组患者股骨内踝负重区、股骨内踝非负重区、股骨外踝负重区、股骨外踝非负重区、胫骨外侧平台区、胫骨内侧平台区、髌后软骨区磁共振T1ρ值明显高于轻度退变亚组患者(P<0.05);磁共振T1rho序列在膝关节早期软骨退变中早期诊断及严重程度评估中的的诊断ROC值及特异度明显高于T2 mapping技术(P<0.05),但后者具有更高的敏感度(P<0.05)。结论磁共振T1rho序列、T2 mapping技术均能有效反映膝关节早期软骨退变中软骨组织学成分变化情况,还可为膝关节软骨退变严重程度评估提供客观依据,二者具有一定互补价值。

基于纳米孔靶向全基因测序技术的新冠肺炎病例快速鉴定研究

目的探索快速识别新型冠状病毒的方法,为新冠肺炎防控提供技术支撑。方法分别运用基于三代纳米孔测序技术平台MinION Mk1C和基于二代测序技术平台Ion Torrent S5的新型冠状病毒(SARS-CoV-2)靶向全基因组测序技术对1例境外输入新冠肺炎确诊病例进行基因组测序。使用artic-ncov2019软件、CLC Genomics Workbench(Version 21.0)软件和DNAstar软件等进行数据处理和分析。结果基于三代纳米孔测序技术平台MinION Mk1C和基于二代测序技术平台Ion Torrent S5分别在7 h和30 h左右获得SARS-CoV-2全基因组数据,分别为29848bp和29801bp,两者共同29801bp数据部分同源性100%,经分析为新冠病毒B.1.1.7变异株。结论在对该病例样本的全基因测序中,基于三代纳米孔测序技术平台MinION Mk1C的SARS-CoV-2靶向全基因组测序技术将检测周期缩短至7 h左右,能够实现快速、准确地对SARS-CoV-2进行实时测序。

利用HiFi读数和k-mer分布特征的序列组装方法

序列组装是利用测序技术得到的读数(read/序列片段)恢复完整基因组序列的方法.当前,第三代测序技术,如单分子实时(SMRT)测序技术和牛津纳米孔技术(ONT),能够产生长度超过10kbp的读数,从而可以解决序列组装中的重复区问题.但是,其测序错误率高达10~15%,使获得完整和准确的基因组序列仍然是一项具有挑战性的工作.最近出现的PacBio HiFi测序技术可以产生长度长(> 10kbp)并且准确性高(> 99.9%)的读数,促进了序列组装领域的研究发展.但是,如何充分利用HiFi读数的优势,开发设计高效的序列组装方法是当前的研究热点.alphaHiASM是一种新的基于HiFi读数和k-mer分布特征的序列组装方法,该方法首先针对HiFi读数长度长和准确性高的优势,设计了一种基于k-mer分布特征的HiFi读数重叠区检测策略.然后,根据上一步检测到的HiFi读数之间的重叠区,以HiFi读数为节点,构建读数重叠图,并提出一种可以衡量重叠区可信程度的边权重计算方法.接着,在该读数重叠图中,抽取路径,形成初始组装结果.最后对初始组装结果进行优化纠错,形成最终结果.该方法在4组不同的真实数据集上和当前流行的组装方法Flye,HiCanu和miniasm进行了性能比较.实验结果表明,alphaHiASM在组装完整性和正确性具有一定的优势.

基于高通量测序技术的9种铁线莲属药材混合粉末分子鉴定

目的建立基于高通量测序技术的9种铁线莲属混合粉末分子鉴定方法。方法采用高通量测序技术,对9种铁线莲属药材混合粉末样品中的总DNA经提取,对ITS2片段进行PCR扩增,利用Illumina MiSeq平台对DNA片段进行双端测序,最后采用FLASH、QIIME、GraPhlAn及MEGA 7.0软件对序列进行整理并聚类分析,鉴定混合粉末中的物种。结果混合粉末样品中得到高质量的ITS2序列共496条,铁线莲属物种含有序列72条,比对出棉团铁线莲、女萎铁线莲、短尾铁线莲、灌木铁线莲、单叶铁线莲、黄花铁线莲、粗齿铁线莲等7个物种,未能比对出东北铁线莲和芹叶铁线莲。结论以ITS2作为DNA条形码,采用高通量测序技术,可以鉴定出铁线莲属药材混合样品中的7个物种,为混合药材的鉴定提供依据。

异时性多复发灶耐药基因分子异质性的胃肠道间质瘤1例报告及文献复习

目的:分析1例长期伊马替尼治疗后多部位、异时性复发病灶敏感/耐药共存的少见胃肠道间质瘤(GISTs)患者的临床资料,揭示GISTs继发突变的异质性是复发耐药过程中的重要属性。方法:收集1例复发GISTs患者的临床资料,采用HE染色法进行组织学观察,免疫组织化学染色检测相关蛋白表达,一代和二代测序技术分析肿瘤基因变异情况。结果:患者,男性,66岁,因胃间质瘤行胃部分切除术。一代测序技术检测结果显示为原癌基因,受体酪氨酸激酶(KIT)基因11号外显子缺失突变(p.551-554del)。伊马替尼治疗1年左右,停药4个月后复发,患者出现脾区病灶,继续采用伊马替尼一线治疗,脾区病灶得到控制。59个月后盆腔发现新病灶,采用伊马替尼加量(600 mg·d^(-1))并舒尼替尼二线治疗,治疗2个月后CT检查结果显示患者脾区病灶大小趋于稳定,而盆腔新病灶体积持续增大。二代测序技术检测结果显示两复发灶在原有KIT基因11号外显子缺失突变的基础上,分别继发KIT基因13号外显子(p.V654A)和17号外显子(p.Y823D)突变。结论:GISTs在靶向药物选择作用的压力下,肿瘤生物学行为表现出复杂性,即时间异质性、空间异型性和分子异质性,不同复发灶可以出现不同的耐药机制。

块菌贮藏期间细菌菌相分析及精油抑菌研究

为揭示印度块菌(Tuber indicum)4℃贮藏期间的表面微生物变化规律,鉴定优势腐败细菌并选择出抑菌效果好的天然抑菌剂。本文利用高通量测序与传统培养技术,对印度块菌贮藏过程中表面菌群结构及优势腐败菌进行研究。通过测定抑菌率,计算半数有效浓度(EC50)等,研究5种天然植物精油对优势腐败菌的抑制效果。结果表明,贮藏期间印度块菌表面菌落总数呈增加趋势,第15 d时达到2.28×10^(6) lg CFU/g并出现异味、发粘或发霉等现象,至30 d时到达货架期终点。贮藏期间的优势菌门为Proteobacteria、Bacteroidetes、Actinobacteria、Firmicutes和Verrucomicrobia。优势菌属为Pseudomonas、Rhizobium、Pedobacter、Sphingobacterium、Flavobacterium、Serratia、Janthinobacterium、Sphingopyxis和Variovorax。此次共分离得到2株可导致块菌腐烂率达90%以上的细菌,经鉴定分别为枯草芽孢杆菌(Bacillus subtilis)和粪小短杆菌(Brachybacterium faecium)。所用5种精油中,柠檬草精油对B.subtilis的抑菌效果最好,EC50值为5.471μL/L;百里香精油对B.faecium的抑菌效果最好,EC50值为6.350μL/L。本研究为后续开展柠檬草和百里香精油复配在块菌采后贮藏保鲜上的应用,延长其货架期提供了一定理论依据。

多发伤患者早期肠道菌群及其代谢产物氧化三甲胺的变化

目的:多发伤患者一旦并发脓毒症和多器官功能障碍综合征则预后不良。本研究通过观察多发伤患者早期肠道菌群及其代谢产物氧化三甲胺的变化,探讨多发伤患者并发脓毒症和多器官功能障碍综合征的可能机制。方法:选取2023年4月至9月江苏大学附属医院收治的符合纳入标准的多发伤患者25例作为多发伤组,选取同期本院体检中心符合纳入标准的健康志愿者25例作为对照组。收集多发伤组伤后1周左右的粪便标本和血浆标本,同期收集对照组体检当天的粪便标本和血浆标本。通过16S rRNA高通量测序技术检测粪便标本,分析多发伤组和对照组肠道菌群的差异。采用高效液相色谱-质谱串联技术检测血浆标本,分析多发伤组和对照组血浆氧化三甲胺水平的差异。结果:多发伤组和对照组的肠道菌群构成存在明显差异。在菌门水平上,多发伤组变形菌门的相对丰度明显高于对照组(P<0.05);厚壁菌门和拟杆菌门的相对丰度明显低于对照组(P<0.05)。在菌属水平上,多发伤组大肠埃希菌属、致病肠球菌属、梭菌属、考拉杆菌属的相对丰度均明显高于对照组(均P<0.05),普氏菌属、双歧杆菌属、瘤胃菌属、乳杆菌属、粪球菌属、布劳特菌属、丁酸弧菌属的相对丰度均明显低于对照组(均P<0.05)。多发伤组血浆氧化三甲胺水平明显高于对照组(P<0.05)。结论:与健康人群相比,多发伤患者早期肠道菌群已发生变化,专性厌氧菌相对丰度下降,兼性厌氧菌相对丰度上升,有益共生菌相对丰度减少,机会致病菌相对丰度增加,这可能与多发伤患者并发脓毒症有关。血浆氧化三甲胺的水平升高可能与肠道菌群的变化和多发伤患者并发多器官功能障碍综合征有关。

不同连作年限设施草莓土壤微生物群落结构分析

为探索不同连作年限设施草莓土壤细菌和真菌群落结构变化及其与土壤环境因子的关系,通过高通量测序对新茬(S1)、连作5年(S5)和连作8年(S8)的设施草莓土壤样品进行测序分析,并测定土壤的理化性质以及土壤酶活性。结果显示,随着设施草莓连作年限的增加,土壤的pH和电导率极显著升高,硝态氮含量极显著降低,有效磷及速效钾的含量先显著减少后显著增加,碱性磷酸酶和脲酶活性呈显著上升后显著降低趋势。随着设施草莓连作年限的增加,土壤细菌ACE指数、Chao1指数、Simpson指数及Shannon指数皆显著增加,而土壤真菌的Alpha多样性指数的变化不显著。土壤中细菌和真菌优势菌群组成发生改变,随着设施草莓连作年限增加,土壤细菌变形菌门、酸酐菌门、硝化螺旋菌门和Latescibacteria相对丰度显著增加,厚壁菌门、放线菌门和拟杆菌门的相对丰度显著降低;在属水平上,设施草莓土壤硝化螺菌属相对丰度显著增加,RB41和芽孢杆菌属相对丰度显著降低,鞘氨醇单胞菌属相对丰度呈先显著升高后显著降低趋势。设施草莓连作土壤优势真菌在门水平无显著变化,在属水平上,土壤真菌占比最高优势菌由毛葡孢属转变为镰孢菌属,草莓土壤病原菌镰孢菌属、曲霉属、枝孢属相对丰度显著增加,乌氏霉属、链格孢属整体增加。相关性分析显示,影响土壤微生物群落的主要因素有土壤pH、电导率、有机质、硝态氮、有效磷和速效钾,微生物群落结构的变化对碱性磷酸酶活性影响较大。