Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), Unidad de Entomología UJI-IVIA. Carretera de Moncada-Náquera Km. 4,5, 46113 Moncada, Valencia, Spain.
J Econ Entomol. 2020 Jun 6;113(3):1471-1478. doi: 10.1093/jee/toaa042.
The development of polymerase chain reaction (PCR) markers to identify the Y chromosome of Ceratitis capitata Wiedemann has permitted the detection of sperm transferred to females during mating. However, a molecular technique to quantify the sperm transferred has not yet become available. The current method to quantify the amount of sperm has been the direct counting of sperm heads. Thus, the purpose of this research was to develop and validate an accurate molecular method of diagnosis based on the application of an absolute quantitative real-time PCR, which allows the assessment of the quantity of sperm stored in the spermathecae. For this, Y-specific sequences were used to re-design and test distinct sperm markers. From the amplification product of samples detected as strong positives in conventional PCR, a cloning process of the target sequence was carried out to build the required standard curve. A series of known dilutions of this standard material was prepared for the absolute quantification process. A Roche Lightcycler 480 Real-Time PCR System and SYBRGreen fluorescent dye were used to quantify the sperm contained in the spermathecae of 4-d-old mated females and virgins. Wild-type and Vienna-8 strain sterile males were used to quantify the sperm transferred at four mating durations (10, 30, 60, and 90 min) under laboratory conditions. To validate the reported quantitative method, our results were compared by counting sperm heads under a fluorescent microscope using the same experimental design. In addition, DNA samples were also evaluated and compared by conventional PCR.
聚合酶链反应（PCR）标记物的发展可用于鉴定黑腹果蝇的 Y 染色体，从而可以检测到交配过程中雄性向雌性转移的精子。然而，目前还没有可用于定量转移精子的分子技术。目前，定量精子数量的方法一直是直接计数精子头。因此，本研究旨在开发和验证一种基于应用绝对定量实时 PCR 的准确分子诊断方法，该方法可以评估储存在精囊中的精子数量。为此，使用 Y 染色体特异性序列重新设计和测试了不同的精子标记物。从在常规 PCR 中被检测为强阳性的样本的扩增产物中，进行了目标序列的克隆过程，以构建所需的标准曲线。为了进行绝对定量过程，制备了一系列已知稀释度的这种标准材料。罗氏 Lightcycler 480 实时 PCR 系统和 SYBRGreen 荧光染料用于定量 4 天龄交配雌性和处女的精囊中所含的精子。使用野生型和维也纳-8 型无菌雄性，在实验室条件下，在四个交配持续时间（10、30、60 和 90 分钟）下定量转移的精子。为了验证报告的定量方法，我们使用相同的实验设计，通过在荧光显微镜下计数精子头来比较结果。此外，还通过常规 PCR 评估和比较了 DNA 样本。
