摘要
家蚕微粒子病被列为蚕种生产的唯一法定检疫对象,母蛾镜检家蚕微孢子虫(Nosema bombycis,Nb)是主要的检疫方法,但其准确性受到多种因素影响。为探究应用荧光增白剂28(Fluorescent Brightener 28,FB28)和碘化丙啶(Propidium Iodide,PI)染色识别Nb孢子的可行性,以感染Nb的家蚕幼虫中肠、蚕卵、母蛾、蚁蚕为材料,联合应用以上2种荧光染色剂进行Nb孢子检测效果试验。在荧光显微镜下可见成熟的Nb孢子被FB28染成蓝色且细胞核被PI染成红色,而未成熟的孢子只能被PI染色。采用2种荧光染色剂染色,在被感染的幼虫中肠、蚕卵、母蛾、蚁蚕中都能够检测到Nb孢子,并且带毒母蛾研磨液样品稀释1 000倍依然可以检测到Nb孢子。幼虫中肠感染后1~2 d很难观察到Nb孢子,感染后3 d可以观察到局部存在少量未成熟的Nb,感染后4 d开始局部出现成熟的Nb,至感染后5~6 d可见组织中大面积分布有成熟孢子和未成熟孢子。荧光显微镜下观察被染色的Nb孢子的细胞核明显小于幼虫中肠细胞核,且亮度明显较强,卵壳、母蛾、蚁蚕表面的几丁质碎片形状与Nb孢子明显不同,并且不能被PI染色。试验结果表明,该染色检测方法可以排除蚕体组织细胞、几丁质碎片的干扰,提高对Nb检测的准确度,并且在Nb浓度较低的情况下,可以提高检测的灵敏度。
Pebrine disease is listed as the only stipulated quarantine objective in silkworm （Bombyx mori） eggs production. To date, microscopic check to female silkworm moths against Nosema bombycis （Nb） has been the main inspection method in practice. However, its accuracy is affected by many factors. In order to explore the feasibility of using Fluorescent Brightener 28 （FB28） and Propidium Iodide （PI） to identify Nb spores, we took the Nb-infected midguts of silk- worm larvae, eggs, female moths and newly-hatched larvae as experimental materials, and conducted effectiveness test on using these two fluorescent staining agents to detect Nb spores. Under fluorescent microscope, the mature Nb spores were stained into blue by FB28 and their nuclei were stained into red by PI, but the immature spores could only be stained by PI. Nb spores could be stained and discriminated in grinding fluid of infected midguts of larvae, eggs, female moths and newly-hatched larvae by using these two fluorescent staining agents. Moreover, Nb spores could also be detected in infected female moth sample after being diluted 1 000 times. Nb spores were difficult to be observed from 1 d to 2 d after larval midguts were infected. At 3 d, a few immature Nb spores could be observed at some locations. At 4 d, mature Nb spores emerged at some locations. From 5 d to 6 d, a great many mature and immature Nb spores were distributed in most locations of the tissue. Under fluorescent microscope, the stained nuclei of Nb spores were obviously smaller than the stained nuclei of larval midgut cells and the former was much brighter than the latter. The chitin debris of silkworm eggs, moths and newly-hatched larvae were obviously different with Nb spores. Moreover, the chitin debris could not be stained by PI. It is concluded that this staining method can eliminate distractions of silkworm tissues/cells and chitin debris, and thus improve the accuracy of inspection. Besides, sensitivity of the detection can be improved in the case of low Nb concentration.
出处
《蚕业科学》
CAS
CSCD
北大核心
2017年第1期62-67,共6页
ACTA SERICOLOGICA SINICA
基金
现代农业产业技术体系建设专项(No.CARS-22)
关键词
家蚕微孢子虫
检疫方法
荧光增白剂28
碘化丙啶
荧光染色
Nosema bombycis
Quarantine method
Fluorescent Brightener 28
Propidium Iodide
Fluorescent staining
