一种血液PCR快速鉴定中华鳖性别方法的优化  

A Rapid Method for Sex Identification in the Chinese Soft-Shelled Turtle Pelodiscus sinensis Based on Direct Blood PCR

作　　者：胡鉴 武志亿 方瑜 余姚辉 李彩燕[1] 宋伟[1,2] HU Jian;WU Zhi-Yi;FANG Yu;YU Yao-Hui;LI Cai-Yan;SONG Wei(College of Biological&Environmental Sciences,Zhejiang Wanli University,Ningbo 315100;College of Animal Sciences,Xinjiang Agricultural University,Urumqi 830000,China)

机构地区：[1]浙江万里学院生物与环境学院,宁波315100 [2]新疆农业大学动物科学学院,乌鲁木齐830000

出　　处：《动物学杂志》2025年第1期136-144,共9页Chinese Journal of Zoology

基　　金：浙江省农业(水产)新品种选育重大科技专项(No.2021c02069-8-3);宁波市科技创新2025重大专项(No.2021Z009)。

摘　　要：目前判断中华鳖(Pelodiscus sinensis)性别的方法主要有外形观察、生殖器官解剖观察和DNA分子鉴定,但这些方法各有局限,不能完全满足生产上的需求,因此急需建立一种快速、损伤小且可准确鉴定中华鳖性别的方法。本研究以中华鳖成鳖为对象,裙边静脉采血,血液经碱煮沸法快速处理后,取上清液作为DNA模板用PTPN11引物进行PCR反应与琼脂糖凝胶电泳,雌性(ZW型)呈现分子量大小为860 bp和1500 bp左右的清晰双带,雄性(ZZ型)呈现分子量大小为860 bp左右的清晰单带。通过外部形态观察和解剖后生殖器官观察对比表明该方法可行,性别鉴定准确率为100%。因此,基于碱煮沸法快速处理的血液直接PCR可以作为一种快速、经济检测中华鳖性别的方法。[Objectives]Currently,the primary methods for determining the sex of Chinese Soft-shelled Turtle Pelodiscus sinensis include visual inspection of external features,examination of reproductive organs after dissection,and DNA PCR analysis.However,each of these methods has its limitations and cannot fully meet the requirements for sex determination in the huge production of Chinese Soft-shelled Turtle.Therefore,there is an urgent need to develop a rapid,minimally invasive and accurate method for determining the genetic sex of Chinese Soft-shelled Turtles.[Methods]In this study,blood was collected from veins of the calipash in adult Chinese Soft-shelled Turtles.The blood supernatant was used as a DNA template for PCR reaction and identification following rapid pretreatment.The feasibility of the genetic method was assessed by optimizing the pretreatment method,blood volume and PCR system(including primer concentration,template concentration,DNA polymerase mix volume and annealing temperature,among other factors),as well as by comparative blood DNA PCR tests,external morphological observations,anddissection of reproductive organs.[Results]The results demonstrated that after rapid pretreatment of 5μl fresh turtle blood using an alkaline boiling method,a 20μl PCR system was prepared by adding 2μl of supernatant DNA template,10μl of DNA polymerase mixture,0.8μl each of upstream and downstream primers,and 6.4μl of ddH2O.PCR amplification was then performed at an annealing temperature of 58℃,followed by detection using 1%agarose gel electrophoresis.The results revealed that female turtles(ZW type)exhibited distinct double bands with molecular sizes approximately 860 bp and 1500 bp,while male turtles(ZZ type)exhibited a clear single band with molecular sizes around 860 bp(Fig.1).The accuracy of sex identification in Chinese Soft-shelled Turtles reached 100%,which was confirmed by the blood DNA PCR validation test,external morphological observations and examinations of reproductive organ(Figs.8﹣10).[Conclusion]The

关 键 词：中华鳖 血液 非损害性取样 PCR 遗传性别鉴定 

分 类 号：Q955[生物学—动物学]