Multiplex PCR for sexing Schistosoma japonicum cercariae and its utility
Multiplex PCR for sexing Schistosoma japonicum cercariae and its utility
复制标题
日本血吸虫尾蚴性别鉴定的多重PCR及其应用
DOI：
10.1007/s00436-019-06431-6
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发表时间：
2019
影响因子：
2
通讯作者：
Xia Chaoming
中科院分区：
文献类型：
--
作者：
Xu Jing;Li Chunxiang;Duan Zhongliang;Yu Dan;Zhang Tingting;Ma Huihui;Wang Xiaoli;Zhan Tingzheng;Xia Chaoming
准确判别日本血吸虫尾蚴性别对于建立单性感染动物模型、规范真实感染强度具有重要意义。在本研究中，建立了一种由两对引物组成的多重PCR技术来确定日本血吸虫尾蚴的性别，其中一对引物扩增W染色体特异的185 bp条带，另一对扩增Z染色体特异的420 bp条带。对于雄性尾蚴 (ZZ)，预计有一个 420 bp 条带，对于雌性尾蚴 (ZW)，可以观察到两个不同的 185 bp 和 420 bp 条带。与曼氏沙门氏菌、埃及沙门氏菌、华支睾吸虫、卫氏并殖吸虫、旋毛虫无交叉反应。对从一只蜗牛逃逸的尾蚴进行性别鉴定后，A组小鼠感染了60只雄性尾蚴，B组小鼠感染了40只雌性尾蚴。同时，C组小鼠感染10只雄性尾蚴和10只雌性尾蚴，并通过多重PCR确定性别。感染后45天，回收雄性和雌性成虫，以验证多重PCR对日本血吸虫尾蚴性别鉴定的准确性，并计算雄性和雌性存活率和配对比率。我们的结果表明，多重PCR技术可以100%准确地区分雄性尾蚴。然而，有时多重PCR的判别结果将混合性尾蚴误判为雌性尾蚴。 C组雄性成虫平均负荷为10.7±2.4，雌性成虫平均负荷为7.7±2.5。 C组雄虫负荷与雌虫负荷差异显着，P值为0.013。 C组真实成虫率为74.2%(95%CI 56.6~91.8%)。这些结果表明，尾蚴感染性别比例平衡的小鼠模型中性别比例偏向雄性，正如我们的多重 PCR 技术所预测的那样。总之，我们的多重PCR技术是区分日本血吸虫尾蚴性别的有效工具，特别是区分雄性尾蚴，对于建立单性尾蚴感染小鼠模型以收获雄性成虫用于抗血吸虫药物筛选具有重要价值。
Accurate discrimination of the Schistosoma japonicum cercariae gender is very important for establishing monosexual infection animal models and for standardizing the real intensity of infection. In this study, a multiplex PCR technique consisting of two pairs of primers, of which one amplifies a 185-bp band specific for the W chromosome and the other amplifies a 420-bp band for the Z chromosome, was established to sex the S. japonicum cercariae. For male cercariae (ZZ), a single 420-bp band is expected, and for female cercariea (ZW), two distinct 185-bp and 420-bp bands can be observed. There was no cross-reaction with S. mansoni, S. haematobium, Clonorchis sinensis, Paragonimus westermani, and Trichinella spiralis. After sexing the cercariae escaped from a single snail, mice in group A were infected with 60 male cercariae and mice of group B were infected with 40 female cercariae. Meanwhile, mice in group C were infected with 10 male and 10 female cercariae that were sexed by multiplex PCR. At 45 days postinfection, male and female adult worms were recovered to verify the accuracy of multiplex PCR for sexing S. japonicum cercariae and to calculate the male and female survival rate and paired worm ratio. Our results showed that the multiplex PCR technique could distinguish male cercariae with 100% accuracy. However, sometimes the discrimination results of multiplex PCR mis-scored mixed sexual cercariae as female cercariae. The mean male adult worm burden in mice of group C was 10.7 ± 2.4, and the mean female adult worm burden was 7.7 ± 2.5. There was a significant difference between the male worm burden and female worm burden in group C. The P value was 0.013. The real paired worm ratio of group C was 74.2% (95%CI 56.6~91.8%). These results demonstrated a male-biased sex ratio in the mice model with equilibrated sex ratio cercariae infection, as predicted by our multiplex PCR technique. In conclusion, our multiplex PCR technique is an effective tool for sexing S. japonicum cercariae, especially for distinguishing male cercariae, which is of great value for establishing monosexual cercariae infection mice models to harvest male adult worms for anti-schistosomal drug screening.