猪链球菌和猪多杀性巴氏杆菌多重实时荧光PCR方法的建立

【背景】猪链球菌(Streptococcus suis,SS)和猪多杀性巴氏杆菌(Pasteurella multocida,Pm)都是能引起宿主致病的人畜共患病原菌,常出现混合感染,临床诊断上易与猪瘟、猪丹毒等混淆。【目的】快速、有效鉴别猪链球菌病和猪多杀性巴氏杆菌病,建立一种能同时检测2种病原的多重实时荧光定量PCR检测方法。【方法】基于猪链球菌的gdh基因和猪多杀性巴氏杆菌的plpE基因,设计2对特异引物及TaqMan探针,以细菌16S rRNA基因设计通用引物及探针,通过对反应条件优化,建立了一种能同时检测猪链球菌和猪多杀性巴氏杆菌的多重实时荧光定量PCR检测方法。【结果】该方法能够特异性地检测猪链球菌和猪多杀性巴氏杆菌,与细菌分离后的测序结果验证完全一致。此方法对重组质粒标准品的最低检出浓度分别为4.53×10²copies/μL和3.97×10²copies/μL。重复性试验结果显示,该方法的组内和组间变异系数均小于3%。【结论】本实验所建立的方法准确、简便、可靠,能够用于2种病原菌的同时检测,为猪链球菌病和猪多杀性巴氏杆菌病的防治提供了有效的检测工具,具有重要的流行病学意义和临床应用价值。     

荧光重组酶介导等温扩增检测食品中单增李斯特菌

[背景] 单增李斯特菌为肉类及乳制品中常见的食源性致病菌,传统的培养法检测无法满足口岸大批量食品的快速检测要求,建立简便、灵敏、快速及现场可操作的技术至关重要。[目的] 建立快速简便的荧光重组酶介导等温扩增（Recombinase-Aided Amplification,RAA）法检测单增李斯特菌,以适应口岸快速通关及监管的实际需求。[方法] 根据单增李斯特菌hlyA基因保守区设计特异性引物、探针,通过引物两两组合结合探针筛选出扩增效率及灵敏度最佳的引物组合,优化反应温度及引物探针浓度,确定最佳反应条件。将建立的荧光RAA法应用于食品基质及实际样品检测中,同时与国标GB 4789.30-2016进行比对验证。[结果] 单增李斯特菌荧光RAA最佳反应温度为42 ℃,最佳引物、探针终浓度均为400 nmol/L。建立的荧光RAA法特异性强,纯菌灵敏度达到3×10² CFU/mL。加标食品基质牛肉、大西洋鲑鱼及再制干酪LB2增菌只需4 h,即可检测原始浓度分别达到0.3、3、30 CFU/mL的单增李斯特菌。荧光RAA法只需5 min即可观察结果,20-30 min完成扩增,速度及灵敏度明显高于国标法。[结论] 建立的荧光RAA法可用于口岸和其他场所进行单增李斯特菌的快速检测与监控。     

腺相关病毒载体工程毒株精细化纯化

[背景] 大量制备高纯度的重组腺相关病毒（Recombinant Adeno-Associated Virus,rAAV）,是以腺相关病毒（AAV）为投递载体的基因靶向治疗或基因工程药物研发过程中需要解决的重要问题。[目的] 利用层析柱法大量纯化质粒和rAAV细胞培养液,以获得高纯度的rAAV颗粒。[方法] 对组装rAAV的3种质粒用HiPrep^TM10 Sepharose 6FF和HiTrap PlasmidSelect Xtra凝胶层析柱纯化,目的是得到组装AAV的超螺旋质粒DNA,然后对组装rAAV过程中的AAV-293细胞培养方法进行优化,组装成功病毒后,再对收集到的细胞提取液用HiLoad^TM10Q和HiLoad^TM10SP阴阳离子交换层析柱纯化,最后用纯化浓缩后的rAAV感染HT1080细胞,通过流式细胞仪检测荧光表达细胞数,计算浓缩后活病毒感染滴度。[结果] HiPrep和HiTrap层析柱纯化后得到大量高纯度的超螺旋质粒DNA,组装病毒后,用HiLoad柱纯化获得大量高纯度的rAAV颗粒,通过测定,rAAV基因组滴度达到（2.46±0.37）×10¹² TCID₅₀/mL （MOI=1.0×10⁴）。[结论] 通过一系列精细化组装纯化制备出高纯度、高滴度rAAV病毒颗粒。     

SARS冠状病毒实时荧光RT-PCR定量检测

为建立一种快速、准确、特异的SARS病毒RNA定量检测方法,根据复合探针荧光定量分析原理,对SARS病毒核酸进行实时荧光定量RT-PCR检测.借助计算机辅助,对SARS病毒基因靶序列以及检测引物和探针进行了优化筛选;利用体外转录SARS病毒RNA靶序列,对RT-PCR反应的镁离子浓度参数进行了优化;比较Trizol法、磁珠法、Qiagen法、煮沸法等4种方法提取RNA的检测效果,建立了样本处理方法;通过对构建的体外转录靶序列模型的检测,对本方法的灵敏度、特异性、定量线性关系、精确度等进行了评价,并通过对42例临床标本的检测对本方法的检测效果进行了评估. 通过克隆SARS病毒核酸靶序列并进行体外转录,获得了长度约1.2 kb的体外转录RNA靶序列;经优化,荧光RT-PCR反应液中的Mg²⁺浓度以4.0 mmol/L为最好;RNA提取方法采用磁珠法效果较好;本方法的检测灵敏度最低可达5个拷贝的体外转录RNA分子,特异性100%,C_t值的CV值小于5%.对临床确诊的42份SARS病人血清和漱口水标本的检测结果表明,该方法的检出率为79%,与荧光抗体检测法的符合率为70%.上述结果表明,该方法建立的荧光定量RT-PCR技术能够快速准确、特异、敏感地对SARS病毒核酸进行定量分析,为临床SARS冠状病毒RNA的检测提供了新的,更为有效的检测方法.     

一株高效甘蔗内生固氮细菌GXS16的鉴定及其对甘蔗的促生长作用

[背景] 我国甘蔗生产中氮肥过量施用严重,导致生产成本居高不下,充分发挥甘蔗与内生固氮菌的联合固氮作用,减少氮肥施用量,对促进我国甘蔗产业可持续发展具有重要意义。[目的] 筛选优势甘蔗内生固氮菌,对其基本特性、联合固氮效率及促生长功能进行评价。[方法] 从甘蔗根系分离到一株内生固氮菌GXS16,利用乙炔还原法测定固氮酶活性,通过PCR扩增nifH基因确定菌株为固氮菌;通过形态观察、Biolog检测和16S rRNA基因序列分析等对菌株进行分类;通过接种盆栽甘蔗检测菌株的促生长作用,采用¹⁵N同位素稀释法检测菌株相对固氮效率。[结果] 菌株GXS16固氮酶活性为2.42μmol-C₂H₄/（h·mL）,根据菌株培养性状和菌体形态观察、Biolog检测、16S rRNA、nifH、acdS基因序列分析结果,菌株GXS16属于伯克氏菌属（Burkholderia）;菌株GXS16还具有1-氨基环丙烷-1-羧酸脱氨酶（1-Aminocyclopropane-1-Carboxylate Deaminase,ACC）活性及合成生长素吲哚乙酸（Indoleacetic Acid,IAA）、降解无机磷的功能;接种GXS16处理甘蔗植株的株高比对照增长15%以上,干重增长20%以上,¹⁵N同位素测定显示甘蔗根、茎、叶从空气中获得氮的百分比分别为7.69%、15.64%和8.72%,效率显著优于模式菌株G.diazotrophicus PAL5。[结论] Burkholderia sp.GXS16是一株高效甘蔗内生固氮菌,具有良好应用前景。     

猪肠病毒G (EV-G)荧光定量PCR检测方法的建立与四川地区分子流行病学调查

【背景】猪肠病毒G (Enterovirus G,EV-G)在猪群中普遍存在,可引起猪的皮肤损伤、肌肉麻痹、肺炎、发热、腹泻等,也存在无症状感染。【目的】建立检测EV-G的荧光定量PCR (real-time quantitative PCR,RT-qPCR)方法并开展四川地区分子流行病学调查。【方法】根据EV-G的5ʹUTR基因保守序列设计特异性引物,建立可检测EV-G各基因型的RT-qPCR,并评估其灵敏性、特异性和重复性,开展四川地区EV-G的流行调查。【结果】标准品在1.89×10²-1.89×10⁸ copies/μL浓度范围内线性关系良好;在特异性试验中,其他9种猪病毒(猪德尔塔冠状病毒、猪流行性腹泻病毒、传染性胃肠炎病毒、日本乙型脑炎病毒、猪萨佩罗病毒、猪细小病毒、猪圆环病毒、猪伪狂犬病病毒和猪繁殖与呼吸综合征病毒)检测均为阴性;最低检测浓度为1.89×10¹ copies/μL;组内和组间变异系数分别小于1%和2%。检测2013-2021年四川省431份猪腹泻样品,EV-G总体阳性率为31.1%,表明该病毒在四川地区的感染较普遍。随机选取9份四川的EV-G阳性样品扩增VP1基因并测序分析,发现四川EV-G流行的基因型包括G1、G3、G4和G9,但以EV-G1为优势基因型。【结论】本研究建立了一种检测EV-G各基因型的RT-qPCR方法,并初步掌握了四川EV-G的流行现状,为后续深入开展该病毒的研究奠定了基础。     

低拷贝病毒RNA捕获及多重实时荧光定量PCR检测

摘要 目的：为提高COVID-19检测灵敏度,减少新冠患者临床假阴性检测结果,本研究建立了一种SARS-CoV-2低拷贝假病毒RNA捕获方法。方法：利用链霉亲和素磁珠,设计并合成生物素探针捕获SARS-CoV-2假病毒RNA;对磁珠用量、生物素探针浓度和洗脱次数等捕获条件进行了优化;参考WHO发布的序列,针对SARS-CoV-2病毒基因组的ORF1ab 基因和 N 基因序列合成引物和TaqMan探针,对捕获的SARS-CoV-2假病毒RNA进行反转录实时荧光定量PCR检测。采用一步法和分步法检测、单重和多重实时荧光定量PCR检测,并分别比较检测结果。结果：本研究设计的生物素探针能富集到距离探针结合位点至少1.8 K处的序列,在生物素探针浓度12.5 μM,磁珠的工作浓度333.33 μg/mL时,合并RNA的两次洗脱液,对低拷贝病毒RNA的捕获效率最高。研究得到对低拷贝病毒RNA分步法比一步法、多重比单重实时荧光定量PCR检测更灵敏,进行分步法、多重实时荧光定量PCR检测限低于10 copies/μL,组间和重复实验组之间CT值变异系数均小于1%。所有阴性对照样品在检测中均为阴性。结论：本研究优化的链霉亲和素磁珠-生物素探针捕获法是富集SARS-CoV-2低拷贝假病毒RNA的有效方法,其抽提RNA病毒的结果优于一些商业化试剂盒,分步法、多重实时荧光定量PCR对SARS-CoV-2低拷贝假病毒RNA实现了高效检测,这为临床检测低拷贝RNA病毒提供了一种参考方案。     

肠炎沙门氏菌基因工程减毒株的免疫效果及安全性评价

[背景] 相较于灭活疫苗和弱毒疫苗,沙门氏菌（Salmonella）基因工程减毒活疫苗具有的优越性逐渐显现,研究也不断深入。[目的] 探究肠炎沙门氏菌G9菌株的4株基因缺失株G9（ΔhilA）、G9（ΔhilD）、G9（ΔssrABhilA）和G9（ΔssrABhilAhilD）的免疫效果及生物安全性。[方法] 以肠炎沙门氏菌基因缺失菌株G9（ΔhilA）、G9（ΔhilD）、G9（ΔssrABhilA）、G9（ΔssrABhilAhilD）及其亲本菌株G9的最佳免疫剂量接种小鼠后,利用间接ELISA法、流式细胞术、MTT法、小鼠攻毒试验及倾注平板法等对各缺失株的免疫效果和安全性进行评价。[结果] G9（ΔhilD）诱导血清IgG抗体效价最高,G9（ΔhilD）和G9（ΔssrABhilAhilD）产生肠黏膜IgA抗体效价最高,4株缺失菌诱导IL-4、IL-10、IFN-γ、TNF-β、MCP-1细胞因子的能力与亲本株G9差异不显著（P>0.05）,产生的CD4⁺/CD3⁺、CD8⁺/CD3⁺ T细胞比率呈上升趋势,而且G9（ΔssrABhilA）和G9（ΔssrABhilAhilD）最高;G9（ΔssrABhilAhilD）诱发的脾淋巴细胞增殖指数最高;免疫小鼠后4株缺失株对G9攻毒提供的保护率为80%-100%,而且小鼠肝脏、脾脏及小肠绒毛无明显病理变化,对鼠伤寒沙门氏菌攻毒提供的保护率为50%-80%;接种后12 d,小鼠肝脏、脾脏及小肠中定殖的菌株能基本清除,而且在体外能连续稳定传代30代。[结论] G9（ΔhilA）、G9（ΔhilD）、G9（ΔssrABhilA）和G9（ΔssrABhilAhilD）对小鼠的免疫效果和生物安全性均良好,有成为肠炎沙门氏菌基因工程减毒活疫苗的可能。     

实时荧光定量PCR检测金山醋酸乳杆菌的方法与应用

[目的] 建立基于实时荧光定量PCR （RT-qPCR）的金山醋酸乳杆菌特异性检测方法,并将其应用于食醋和白酒发酵过程样品的快速检测。[方法] 筛选金山醋酸乳杆菌基因组中特异性强的基因序列作为模板设计特异性引物,通过标准菌株、醋醅样品进行PCR验证引物的特异性和准确性,建立RT-qPCR方法分析金山醋酸乳杆菌在不同地区酒醅、醋醅和食醋样品中的含量。[结果] 以金山醋酸乳杆菌的苯丙氨酰-tRNA合成酶α亚基（编码pheS基因）为参考序列,设计了一对GC含量55%、T_m值59℃、可扩增199 bp片段的引物。建立的RT-qPCR方法特异性强、灵敏度高且重复性好,检测浓度范围为2.24-10.24 lg （copies/μL）,成功应用于4个地区的酒醅、醋醅和食醋样品检测。对镇江香醋醋酸发酵过程的研究表明,醋醅中的金山醋酸乳杆菌数量先迅速增加,随后稳定在10⁸ copies/g干醅。[结论] 本研究建立的RT-qPCR方法可对食醋和白酒发酵过程中金山醋酸乳杆菌进行特异性鉴定和快速定量。     

新疆野果林木霉分离鉴定、抑菌作用及固态发酵配方优化

[背景] 新疆野苹果病害发生严重,而木霉（Trichoderma spp.）是重要的植物病害生防菌,因此可利用其对新疆野苹果病害进行防治。[目的] 分离鉴定新疆野果林木霉菌株,分析其对新疆野苹果2种病原菌的抑菌能力,为新疆野苹果专用木霉菌肥的制备提供菌种资源。[方法] 利用平板稀释法对新疆野果林4种草本植物根围木霉菌进行分离,通过形态学与分子生物学方法进行菌种鉴定,平板对峙试验检测其抑菌能力并优化木霉菌株的固态发酵配方。[结果] 共分离得到2种木霉菌共42株,分别为34株棘孢木霉（T. asperellum）和8株深绿木霉（T. atroviride）;棘孢木霉TasT01对苹果炭疽病菌（Colletotrichum gloeosporioides,Cgl）和苹果斑点落叶病病原菌（Alternaria alternaria f.sp.mali,Aal）的抑菌率分别为58.60%和57.14%;深绿木霉TatT35对Cgl和Aal的抑菌率分别为43.86%和36.90%。进一步显微镜观察发现,TasT01可通过菌丝缠绕的方式抑制2种病原菌的生长。TatT35菌株固态发酵配方为稻壳90%、麦麸10%、（NH₄）₂SO₄ 0.5%时,孢子浓度达1.0×10⁸个/g。[结论] TasT01和TatT35对新疆野苹果2种病原菌都有很好的抑制效果,固态发酵配方优化后可以提高孢子浓度,该研究结果为新疆野苹果真菌病害的生物防治提供了可借鉴的方法。     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.