Detection and Variability of Aster Yellows Phytoplasma Titer in Its Insect Vector, Macrosteles quadrilineatus (Hemiptera: Cicadellidae)
Detection and Variability of Aster Yellows Phytoplasma Titer in Its Insect Vector, Macrosteles quadrilineatus (Hemiptera: Cicadellidae)
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DOI：
10.1603/ec11183
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发表时间：
2011-12-01
影响因子：
2.2
通讯作者：
Groves, R. L.
中科院分区：
文献类型：
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作者：
Frost, K. E.;Willis, D. K.;Groves, R. L.
紫菀黄化植原体（AYp）是由紫菀叶蝉Macrosteles quadrilineatus福布斯（Forbes）以持续和繁殖的方式传播的。为了研究AYp复制和检查个体紫菀叶蝉中AYp滴度的变异性，我们开发了一种定量实时聚合酶链反应测定昆虫DNA提取物中AYp浓度的方法。通过比较从昆虫全DNA提取物中扩增未知量的AYp靶基因序列延伸因子TU（tuf）与含有已知量的克隆到质粒中的tuf基因序列的稀释系列的扩增，实现了AYp DNA的绝对定量。该方法的能力和局限性进行了评估，通过进行时间过程实验，不同的AYp在紫菀叶蝉的孵化时间从0到9天后，48小时的收购访问期AYp感染的植物。在107只紫菀叶蝉中测量平均AYp滴度，并表示为Log 10（拷贝/昆虫），范围为3.53（+/-0.07）至6.26（+/-0.11），发生在采集访问期后1天和7天。AYp滴度每只昆虫和相对于紫菀叶蝉染色体参考基因，cp 6无翅（cp 6），增加了近100倍，昆虫收购AYp。紫菀叶蝉没有暴露于AYp感染的植物获得的高定量循环值被解释为背景，并用于定义定量实时聚合酶链反应测定的检测限。这种方法将提高我们的能力，研究AYp复制的紫菀叶蝉的生物因素，并确定是否AYp滴度与频率的传输。
The aster yellows phytoplasma (AYp) is transmitted by the aster leafhopper, Macrosteles quadrilineatus Forbes, in a persistent and propagative manner. To study AYp replication and examine the variability of AYp titer in individual aster leafhoppers, we developed a quantitative real-time polymerase chain reaction assay to measure AYp concentration in insect DNA extracts. Absolute quanti_cation of AYp DNA was achieved by comparing the amplification of unknown amounts of an AYp target gene sequence, elongation factor TU (tuf), from whole insect DNA extractions, to the amplification of a dilution series containing known quantities of the tuf gene sequence cloned into a plasmid. The capabilities and limitations of this method were assessed by conducting time course experiments that varied the incubation time of AYp in the aster leafhopper from 0 to 9 d after a 48 h acquisition access period on an AYp-infected plant. Average AYp titer was measured in 107 aster leafhoppers and, expressed as Log 10 (copies/insect), ranged from 3.53 (+/- 0.07) to 6.26 (+/- 0.11) occurring at one and 7 d after the acquisition access period. AYp titers per insect and relative to an aster leafhopper chromosomal reference gene, cp6 wingless (cp6), increased approximate to 100-fold in insects that acquired the AYp. High quantification cycle values obtained for aster leafhoppers not exposed to an AYp-infected plant were interpreted as background and used to define a limit of detection for the quantitative real-time polymerase chain reaction assay. This method will improve our ability to study biological factors governing AYp replication in the aster leafhopper and determine if AYp titer is associated with frequency of transmission.