Efficacy of fluorescent calcein mark retention and identification in endangered Colorado pikeminnow Ptychocheilus lucius

Distinguishing hatchery‐reared fishes from wild conspecifics can be required to quantify the success of augmentation programmes. This study estimated the probability of identifying calcein‐marked, hatchery‐reared Colorado pikeminnow Ptychocheilus lucius Girard from external and internal structures. Both control and marked fish held in the laboratory were correctly identified 80% of the time after 300 days. A fluorometer was more accurate 180 days post‐marking, whereas visual observation was more discriminating at 300 days. There were no differences in detection rates among structures in laboratory fish, and for most structures of fish held outdoors, detection rates were <20%. Overall, a strong negative effect of light on mark persistence was observed. Also, an initial positive effect of fish size on mark fluorescence and a negative effect of fish growth were detected. These results suggest the potential use of calcein as a batch‐marking tool would be aided by future studies that better quantify light exposure and calcein mark persistence in both field and experimental settings.     

Musculoskeletal responses of 2-year-old Thoroughbred horses to early training. 6. Bone parameters in the third metacarpal and third metatarsal bones

AIM: To determine the effect of a known training regimen on the size and mineral content of the third metacarpal (Mc3) and third metatarsal (Mt3) bones of 2-year-old Thoroughbred horses trained on racetracks.

METHODS: Mc3 and Mt3 of seven horses trained on grass and sand tracks were scanned at several sites using conventional quantitative and peripheral computed tomography (CT). Bone dimensions and density in the diaphysis and epiphysis were compared with those from seven untrained horses. Calcein label was injected in two clusters, during Weeks 9 and 12. The extent and rate of diaphyseal modelling was determined by confocal fl uorescent microscopic examination of thin plane parallel sections of the mid-metacarpal region.

RESULTS: Volumetric bone mineral density (BMDv) of the epiphysis was markedly higher and of the diaphysis was slightly higher in trained compared with untrained horses, but greater bone size in the trained horses had the greatest effect on an index of bone strength. Active osteons, defined as Haversian systems containing calcein label, were fewer, of smaller diameter at the time of calcein injection, and had a greater bone apposition rate in trained than in untrained horses.

CONCLUSIONS: Conventional training of 2-year-old Thoroughbred racehorses over a 13-week period had a significant effect on bone size, density and strength index when compared to untrained horses.

CLINICAL RELEVANCE: Bone responded rapidly to early training. The data provide reference values and sites for use in longitudinal studies of commercial training regimens.     

马铃薯S病毒PVSO株系RT-LAMP检测方法的建立及应用

为建立一种马铃薯S病毒普通株系PVSO快速、灵敏的RT-LAMP可视化检测方法。根据马铃薯S病毒普通株系的CP核苷酸序列设计4条引物,以感染PVS的马铃薯叶片总RNA为模板,采用一步法RT-LAMP,在62 ℃下反应60 min,扩增产物利用琼脂糖电泳分析和钙黄绿素染料显色判断结果,同时对其特异性和灵敏性进行测定。结果表明,建立的RT-LAMP方法可特异地对PVSO进行检测,检测灵敏度为RT-PCR的100倍。对33份马铃薯样品检测表明,RT-LAMP与RT-PCR方法检测结果基本一致。因此建立的PVSO的 RT-LAMP可视化检测方法简便,特异性强,灵敏度高,适合PVSO的快速检测。     

拌饲投喂钙黄绿素对中华倒刺鲃幼鱼不同组织抗氧化酶活性和脂质过氧化的影响

为探究拌饲投喂方式下,钙黄绿素(calcein, CAL)对中华倒刺鲃幼鱼不同组织抗氧化水平和脂质过氧化的影响,以及正常投喂后的恢复情况,并进一步评价CAL拌饲投喂的毒性效应,实验将含有不同剂量CAL(0、2、8和32 g/kg)的饲料连续投喂中华倒刺鲃幼鱼16 d(即毒性积累实验),随后使用不含CAL的饲料投喂暂养32 d(即毒性消除实验)。毒性积累实验结果显示,在CAL拌饲投喂剂量 ≤ 32 g/kg的情况下,当累积投喂时间 ≤ 8 d时,过量的活性氧(ROS)能被中华倒刺鲃幼鱼的抗氧化系统成功清除,但更长的投喂时间(如16 d)可能产生一定的毒性效应,并导致鱼体肝胰脏和肾脏组织的氧化应激过度。毒性消除实验结果显示,经32 d正常投喂后,中华倒刺鲃幼鱼血清、肝胰脏和肾脏组织的大部分抗氧化酶指标、非酶抗氧化物、脂质过氧化产物基本恢复至安全水平。研究表明,当拌饲投喂剂量 ≤ 32 g/kg时,CAL对中华倒刺鲃幼鱼的毒性作用与累积投喂时间有着密切关系,且当使用 ≤ 32 g/kg的CAL拌饲剂量投喂标记中华倒刺鲃幼鱼时,CAL的累积投喂时间应控制在8 d以内,标记后的幼鱼应当至少暂养32 d。研究结果对CAL在鱼类荧光标记中的安全有效使用具有重要的指导意义。     

鹅新城疫病毒RT-LAMP可视化检测方法的建立

本研究拟建立一种能在基层实时、便捷诊断鹅新城疫病毒（goose Newcastle disease virus，GNDV）的检测方法。根据GenBank中公布的GNDV F基因的高度同源保守序列设计特异性引物，并在反应体系中添加钙黄绿素/氯化锰指示剂，建立可视化RT-LAMP检测方法。结果显示，建立的方法能特异地检测出GNDV及NDV Lasota疫苗株，而小鹅瘟病毒（GPV）、禽流感病毒（AIV）、鸭坦布苏病毒（DTMUV）等其他病毒均为阴性。所建立的GNDV可视化RT-LAMP检测方法对RNA的最低检测限为10 pg，反应过程不需PCR仪等复杂的仪器，50 min即可完成反应，可经肉眼观察反应体系颜色判定结果。该方法特异性强、灵敏度高，且操作安全、简便、快捷，可满足基层筛查GNDV的需求。     

茜素红与钙黄绿素标记不同规格黑鲷幼鱼的比较研究

采用茜素红(ARS)和钙黄绿素(CAL)以浸染方式对大、小2种规格的黑鲷(Acanthopagrus schlegelii)幼鱼进行荧光标记技术研究。急性染色实验结果显示,2种染料对2种规格黑鲷幼鱼成活率无显著影响;后续养殖实验结果表明,两者对2种规格黑鲷幼鱼生长率亦无显著影响。ARS和CAL对黑鲷幼鱼硬组织的标记效果随着染料浓度的增加而提高,ARS标记效果优于CAL。比较2种规格的黑鲷幼鱼荧光标记结果,ARS未见显著性差异,CAL小规格标记效果优于大规格。比较各取样组织标记效果,ARS对鳍棘标记取得了最好的标记效果,鳞片和鳍条次之,耳石标记效果最差;CAL对鳍棘、鳍条、鳞片的标记效果相似或等级相同,耳石次之。ARS标志技术更具应用和研究价值。     

基于荧光显色的IBV LAMP检测方法研究

【目的】建立基于荧光显色的可视化禽传染性支气管炎病毒（IBV）环介导等温扩增（LAMP）检测体系,为IBV的快速临床检测提供有力的技术支持。【方法】根据NCBI中收录的IBV 5a+5b基因的8个区域设计了3对LAMP引物,通过对反应体系各组分和条件的优化,建立IBV LAMP检测方法,并对该方法的特异性和敏感性进行验证。分别用SYBR Green I和一定浓度比的钙黄绿素/锰离子混合物作为显色剂,对IBV LAMP检测结果进行可视化判定。应用该检测体系和PCR方法对临床送检的60份样品同时进行检测,比较2种方法的阳性检出率。【结果】成功建立了IBV LAMP检测方法,该方法能够在等温（63 ℃）条件下1 h内完成反应,具有良好的敏感性和特异性,最低可以检出1拷贝/μL的阳性重组质粒,比普通PCR方法敏感100倍,而对其他病毒检测结果均为阴性。应用SYBR Green I和钙黄绿素/锰离子显色时,阴阳性样品均有强烈的颜色对比,其中0.05 mmol/L钙黄绿素与0.6 mmol/L锰离子混合液可以用于LAMP扩增产物的判断,其结果与琼脂糖凝胶电泳结果一致。在对60份临床样本的检测中,LAMP和PCR方法检测出的阳性样本数量分别为49份和42份。【结论】建立了基于荧光显色的IBV LAMP检测方法,该方法具有在科研机构、基层实验室特别是检测现场推广和应用的潜力。     

番石榴焦腐病菌LAMP检测方法的建立及应用

 以钙黄绿素（calcein）为指示剂,建立了番石榴焦腐病菌（Botryosphaeria rhodina）的环介导等温扩增（Loop mediated isothermal amplification, LAMP）可视化快速检测方法。在该方法中,以番石榴焦腐病菌特异的核糖体转录间隔区（Internal transcribed spacer, ITS）序列为目的DNA片段,设计4条引物（2条内引物、2条外引物）,优化LAMP反应条件和反应体系,并进行特异性和灵敏度验证。研究确定最佳反应温度为64℃,反应时间1 h;特异性检测结果显示,15株不同地理来源的番石榴焦腐病菌和10份番石榴焦腐病病果组织LAMP产物均为阳性（绿色）,而26株其它病原菌及10份健康番石榴果实组织LAMP产物均为阴性（橘黄色）;灵敏度验证结果显示,该方法的检测灵敏度在DNA水平上可达到10 pg。实验结果表明,快速、准确、灵敏的LAMP检测方法适合基层部门及田间检测番石榴焦腐病菌使用。     

Efficacy of calcein as a chemical marker of green‐lipped mussel (Perna canaliculus) larvae and its potential use for tracking larval dispersal

An optimal chemical shell marking protocol was developed for the New Zealand green‐lipped mussel, Perna canaliculus with a view to its future use in larval tracking experiments. Larval P. canaliculus aged either 10, 15 or 19 days post fertilization were immersed in treatments of 50, 100 and 200 mg L^?1 of calcein for a period of 24 h before measurements of shell mark brightness were taken. There was 100% marking success in all calcein treatments for all age classes, with 19‐day larvae immersed in 200 mg L^?1 calcein producing the brightest mark. Growth was not affected by calcein immersion; however, 10‐day larvae exhibited significantly higher levels of mortality compared with 15‐ and 19‐day larvae suggesting a reduced resilience to the marking protocols in younger larvae. In a mass staining experiment, a solution of 100 mg L^?1 calcein was used to successfully stain15.6 million hatchery reared P. canaliculus larvae. Calcein, therefore, offers a low impact method with which to stain the sensitive early life stages of this species thus providing a rapid method for identifying individuals of interest, i.e. individuals released in the wild or specific family lines within a hatchery environment.     

基于钙黄绿素显色的可视化LAMP检测猪流行性腹泻病毒的研究

针对猪流行性腹泻病毒(PEDV)的NP基因设计1套环介导等温扩增(LAMP)引物,在反应体系中添加钙黄绿素/氯化锰指示剂代替传统的反应后添加SYBR Green Ⅰ染料,建立了基于钙黄绿素的可视化LAMP检测PEDV的方法。该方法能在65 ℃ 1 h内特异性扩增PEDV,与猪瘟病毒(CSFV)、猪传染性胃肠炎病毒(TGEV)、猪轮状病毒(PoRV)及大肠杆菌等均无交叉反应,检测下限为3.73 pg/μL,其结果可用肉眼判断,快捷方便。用该方法对龙岩学院动物医学研究所接诊的93份腹泻病例进行检测,结果表明,LAMP方法检测PEDV的阳性率为43.01%(40/93),高于普通PCR的检出率(38.7%,36/93)。本试验建立的可视化LAMP检测方法能用于PED诊断。