家畜胚胎性别鉴定技术的研究进展

性别控制是按照人们的意愿生产特定性别后代的动物繁殖新技术。早期胚胎性别鉴定是人们实现性别控制的重要途径。随着科学技术的发展和各种新的研究工具在性别鉴定中的应用,尤其是聚合酶链式反应(PCR)技术的应用,性别鉴定在生产实践中的应用越来越广泛。本文综述了胚胎性别鉴定方法的基本原理和研究概况,并阐述了该技术存在的问题和发展前景。     

青稞黑穗病菌LAMP检测体系的建立

【目的】大麦坚黑粉菌(Ustilago hordei)和裸黑粉菌(Ustilago nuda)是引起青藏高原青稞黑穗病的主要病原菌,建立快速、简便、特异性强的大麦坚黑粉菌和裸黑粉菌环介导等温扩增技术(LAMP)检测体系,检测青稞种子的带菌量,再依据带菌程度进行播前种子处理,是控制青稞黑穗病的重要技术手段。【方法】根据大麦坚黑粉菌(Ustilago hordei)和裸黑粉菌(Ustilago nuda)的ITS基因序列与其他对照菌株同源性低的区段设计并获得有效引物1组,建立LAMP检测体系,并选取影响LAMP反应体系的5个主要因素dNTPs、甜菜碱、镁离子、Bst DNA聚合酶及温度进行单因素试验对其进行优化,最后对优化后的LAMP检测体系进行灵敏度和特异性检测。【结果】建立了青稞黑穗病菌的LAMP检测体系,其优化后dNTPs浓度为1.8 mmol/L、甜菜碱浓度为0.8 mmol/L、镁离子浓度为10 mmol/L、Bst DNA聚合酶浓度为320 U/mL,反应温度为65℃。应用该体系在45 min内能检测出2×10~(-4)ng/μL的裸黑粉菌,在60 min内能检测出2×10~(-4) ng/μL的大麦坚黑粉菌,即在60 min内可检测出2×10~(-4)ng/μL的大麦坚黑粉菌和裸黑粉菌。建立的青稞黑穗病菌LAMP检测体系对大麦坚黑粉菌和祼黑粉菌具有特异性。【结论】建立了青稞黑穗病菌的LAMP检测体系,该体系能在1 h内检测出2×10~(-4)ng/μL的裸黑粉菌和大麦坚黑粉菌。     

快速检测大肠杆菌O157的LAMP方法的建立与评价

利用DNA环介导恒温扩增技术(Loop-mediated isothermal amplification,LAMP),以rfbE基因为靶序列,设计LAMP引物,建立LAMP反应体系与条件,进行灵敏度和特异性试验.结果显示,整个检测过程仅需1h即可通过肉眼直接目测试验结果.在灵敏度试验中,rfbE-LAMP对O157菌液和模拟阳性样品的最低检测限分别为1CFU/mL和1 CFU/g.在特异性试验中,以其他血清型大肠杆菌以及沙门杆菌基因组DNA为模板对rfbE基因进行检测,均没有发生非特异性扩增反应.试验建立的LAMP检测方法具有操作简单安全、检测灵敏度高、特异性高的特点,能够满足基层实验室、应急检测或现场监测等方面的使用需求.     

Loop-mediated isothermal amplification (LAMP) test for specific and rapid detection of Brucella abortus in cattle

Background: Brucella abortus, the major causative agent of abortion in cattle and a zoonotic pathogen, needs to be diagnosed at an early stage. Loop-mediated isothermal amplification (LAMP) test is easy to perform and also promising to be adapted at field level.

Objective: To develop a LAMP assay for specific and rapid detection of B. abortus from clinical samples of cattle.

Methods: LAMP primers were designed targeting BruAb2_0168 region using specific software tool and LAMP was optimized. The developed LAMP was tested for its specificity with 3 Brucella spp. and 11 other non-Brucella spp. Sensitivity of the developed LAMP was also carried out with known quantity of DNA. Cattle whole blood samples and aborted fetal stomach contents were collected and used for testing with developed LAMP assay and results were compared with polymerase chain reaction (PCR).

Results: The developed LAMP assay works at 61 °C for 60 min and the detection limit was observed to be 100-fold more than the conventional PCR that is commonly used for diagnosis of B. abortus. Clinical sensitivity and specificity of the developed LAMP assay was 100% when compared with Rose Bengal plate test and standard tube agglutination test. SYB® green dye I was used to visualize the result with naked eye.

Conclusion: The novelty of the developed LAMP assay for specifically detecting B. abortus infection in cattle along with its inherent rapidness and high sensitivity can be employed for detecting this economically important pathogen of cattle at field level as well be exploited for screening of human infections.     

Functional Analysis of Lysosomes During Mouse Preimplantation Embryo Development

Lysosomes are acidic and highly dynamic organelles that are essential for macromolecule degradation and many other cellular functions. However, little is known about lysosomal function during early embryogenesis. Here, we found that the number of lysosomes increased after fertilization. Lysosomes were abundant during mouse preimplantation development until the morula stage, but their numbers decreased slightly in blastocysts. Consistently, the protein expression level of mature cathepsins B and D was high from the one-cell to morula stages but low in the blastocyst stage. One-cell embryos injected with siRNAs targeted to both lysosome-associated membrane protein 1 and 2 (LAMP1 and LAMP2) were developmentally arrested at the two-cell stage. Pharmacological inhibition of lysosomes also caused developmental retardation, resulting in accumulation of lipofuscin. Our findings highlight the functional changes in lysosomes in mouse preimplantation embryos.     

植物罹病组织中象耳豆根结线虫的LAMP快速检测方法

【目的】建立一种基于环介导等温扩增（Loop-mediated isothermal amplification,LAMP）技术,从植物罹病组织中直接检测象耳豆根结线虫（Meloidogyne enterolobii）的快速检测方法,为象耳豆根结线虫的监测和防治提供技术支持。【方法】根据象耳豆根结线虫与其它根结线虫ITS序列差异设计LAMP特异性引物,通过对LAMP反应条件中的Mg2+、dNTPs、甜菜碱和反应时间进行优化,同时对检测体系的特异性和灵敏度进行验证,建立一种从罹病植物组织中检测象耳豆根结线虫的LAMP快速检测方法。【结果】象耳豆根结线虫LAMP检测体系优化结果表明在Mg2+的浓度为5.0 mmol•L-1、dNTPs的浓度为2.4 mmol•L-1、不添加甜菜碱、反应时间为45 min的条件下,扩增效率最优。本研究建立的LAMP检测方法能够从11个植物线虫种群中特异检测出象耳豆根结线虫,检测灵敏度为1/200000头线虫DNA,比普通PCR灵敏100倍,能够直接从植物根结中检测出象耳豆根结线虫,准确度为100%。【结论】本研究以rDNA-ITS序列为靶基因建立象耳豆根结线虫LAMP快速分子检测方法,具有特异性强、灵敏度高、简单、快速、经济等特征,能够从罹病植物组织中快速准确地检测出象耳豆根结线虫,具有极高的实践应用价值。     

环介导等温扩增技术检测酸乳中蜡样芽孢杆菌

目的：建立快速准确检测酸乳中蜡样芽孢杆菌的方法.方法：根据蜡样芽孢杆菌hblA基因设计特异性引物,通过环介导等温扩增技术（LAMP）直接检测酸乳中蜡样芽孢杆菌,扩增产物电泳检测.结果：LAMP法检测酸乳中蜡样芽孢杆菌的特异性强,方法检出限6.4CFU/mL,人工污染检出限21CFU/mL.结论：LAMP法用于检测酸乳中蜡样芽孢杆菌的灵敏度高、耗时短,为酸乳中蜡样芽孢杆菌的快速检测提供了新的方法.     

柑橘溃疡病菌的普通LAMP及快速LAMP检测方法的建立

建立柑橘溃疡病菌的普通LAMP和快速LAMP检测方法,使其能应用于基层检验检疫部门对病害的快速检测.利用柑橘溃疡病菌基因组特有的保守区域设计LAMP引物,通过优化反应条件,建立柑橘溃疡病菌的普通LAMP检测体系;在普通LAMP引物的基础上设计一对环引物,建立柑橘溃疡病菌的快速LAMP检测体系,并以多种参比菌DNA以及健康柑橘叶片基因组DNA为模板对普通LAMP和快速LAMP检测体系的特异性进行了验证,利用柑橘溃疡病菌菌液和DNA溶液梯度稀释液对普通LAMP和快速LAMP检测体系的灵敏度进行了验证.普通LAMP检测体系菌体和DNA检测灵敏度分别达到了2.25×104 cfu和2.03×10-1 ng,快速LAMP检测体系菌体和DNA检测灵敏度分别达到了2.25 cfu和2.03×10-5ng.在特异性测试中,普通LAMP检测体系与快速LAMP检测体系均仅对柑橘溃疡病菌进行扩增,对非靶标菌和柑橘叶片基因组DNA不产生扩增,普通LAMP与快速LAMP检测体系特异性测试结果一致.快速LAMP检测体系在0.5h内就可以达到普通LAMP检测体系的扩增量,是普通LAMP检测体系反应时间的一半,大大提高了检测的效率;快速LAMP检测体系菌悬液和DNA检测灵敏度均比普通LAMP检测体系提高了10 000倍.成功地建立了柑橘溃疡病菌的普通LAMP及快速LAMP检测方法,为柑橘溃疡病菌的检测提供了一种新的简便、快速的检测手段.     

检测猪繁殖与呼吸综合征病毒和猪圆环病毒的二重荧光LAMP方法的建立

建立一种二重荧光LAMP的检测猪繁殖与呼吸综合征病毒(PRRSV)和猪圆环病毒(PCV)的快速诊断技术。根据已发表的PRRSV NSP2基因和PCV2 ORF2基因序列保守区域,分别设计并合成了两组针对PRRSV和PCV2序列的特异性引物和两条探针,两条探针分别标记不同的荧光基团,PRRSV-Probe 5’端标记FAM荧光基团,3’端标记BHQ1淬灭基团,PCV-Probe 5’端标记CY5.5荧光基团,3’端标记BHQ2淬灭基团。对组成反应体系中的引物、探针以及反应试剂进行优化,建立了二重荧光LAMP检测PRRSV和PCV2方法。对建立的方法进行特异性和敏感性测试,并用模拟混合样品和临床样品进行检测验证。结果显示,本研究建立的方法可以鉴别区分PRRSV和PCV2,特异性试验表明该方法只检测出PRRSV或PCV2,且与参试的对照病毒无交叉反应,特异性好。敏感性测试表明该方法最低检测PRRSV或PCV为100拷贝,敏感性好。对临床样品的检测,结果与荧光定量PCR方法检测结果一致。本研究建立的PRRSV和PCV2二重荧光LAMP检测方法,具有良好的特异性和敏感性,可用于临床样品中检测PRRSV和PCV2。     

草鱼呼肠孤病毒逆转录环介导等温扩增(RT-LAMP)检测方法的建立

根据草鱼呼肠孤病毒(grass carp reovirus,GCRV)衣壳蛋白VP6编码基因的序列设计特异性引物,以病毒全基因组RNA为模板,通过对反应条件进行优化,建立了GCRV的逆转录环介导等温扩增(RT-LAMP)检测方法。检测结果表明,本方法可在63℃下1 h内实现靶片段的大量扩增,扩增产物经凝胶电泳呈现梯型条带,反应体系中添加SYBR Green I荧光染料后,绿色阳性结果明显区别于橙色阴性结果。该检测体系针对草鱼呼肠孤病毒的检测灵敏度高,其最低检测限为33 pg,与常规RT-PCR方法相比较,灵敏度高10倍,且与斑点叉尾鮰呼肠孤病毒(CCRV)、鲤春病毒血症病毒(SVCV)、锦鲤疱疹病毒(KHV)、大鲵虹彩病毒(GSIV)等无交叉反应。该方法灵敏度及特异性高,且不需昂贵仪器设备,为快速检测草鱼呼肠孤病毒与诊断草鱼出血病提供了简捷快速的技术手段。