{"title":"TaqMan探针实时荧光定量PCR技术在养殖环境和鱼类组织中快速检测和定量的建立及应用","authors":"Shu-Quan Guo, Yao-Wu Fu, Ting-Long Hou, Shi-Lu Huang, Qi-Zhong Zhang","doi":"10.1016/j.vetpar.2024.110381","DOIUrl":null,"url":null,"abstract":"<p><p>Ichthyophthirius multifiliis, a pathogenic ciliate, is a crucial pathogen of freshwater fish and can result in severe economic loss in the aquaculture industry worldwide. It is necessary to develop a sensitive and accurate method for detecting I. multifiliis in farming environments and fish skin and gills to protect fishes from infection of the parasite due to a lack of both safe and effective treatment drugs. The present study established a new TaqMan probe-based quantitative PCR (qPCR) detection method targeting the coding region of the cathepsin L cysteine protease (ICP2) gene of I. multifiliis. The sensitivity, specificity, reproducibility and application for detection and diagnosis of the TaqMan probe-based qPCR method were evaluated. In addition, the linear model between the cycle threshold (Ct) and the logarithmic starting quantity (SQ) of the number of theronts per 1 L of sterile water was developed as Ct = -3.312lg(SQ)+ 34.47 with an R<sup>2</sup> of 0.9636 and a minimum detection limit of 4 theronts per 1 L of water and could be employed to determine the theront number based on Ct value. The results of the detection of trial infection samples with the TaqMan probe-based qPCR method showed that the tissues of fish individuals infected with I. multifiliis and the tank water samples were positive detection signals. In contrast, the tissues and water samples from uninfected fish individuals and tanks containing healthy fish showed no signals. The detection results demonstrated the reliability of this detection method. Overall, the novel TaqMan probe-based qPCR method with high sensitivity and specificity as well as repeatability for detection of I. multifiliis was a valuable tool in detecting the parasite in farming water, pond sediments, and fish tissues and could provide early warning for prevention of the disease caused by I. multifiliis.</p>","PeriodicalId":23716,"journal":{"name":"Veterinary parasitology","volume":"334 ","pages":"110381"},"PeriodicalIF":2.0000,"publicationDate":"2024-12-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Establishment and application of TaqMan probe-based quantitative real-time PCR for rapid detection and quantification of Ichthyophthirius multifiliis in farming environments and fish tissues.\",\"authors\":\"Shu-Quan Guo, Yao-Wu Fu, Ting-Long Hou, Shi-Lu Huang, Qi-Zhong Zhang\",\"doi\":\"10.1016/j.vetpar.2024.110381\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Ichthyophthirius multifiliis, a pathogenic ciliate, is a crucial pathogen of freshwater fish and can result in severe economic loss in the aquaculture industry worldwide. It is necessary to develop a sensitive and accurate method for detecting I. multifiliis in farming environments and fish skin and gills to protect fishes from infection of the parasite due to a lack of both safe and effective treatment drugs. The present study established a new TaqMan probe-based quantitative PCR (qPCR) detection method targeting the coding region of the cathepsin L cysteine protease (ICP2) gene of I. multifiliis. The sensitivity, specificity, reproducibility and application for detection and diagnosis of the TaqMan probe-based qPCR method were evaluated. In addition, the linear model between the cycle threshold (Ct) and the logarithmic starting quantity (SQ) of the number of theronts per 1 L of sterile water was developed as Ct = -3.312lg(SQ)+ 34.47 with an R<sup>2</sup> of 0.9636 and a minimum detection limit of 4 theronts per 1 L of water and could be employed to determine the theront number based on Ct value. The results of the detection of trial infection samples with the TaqMan probe-based qPCR method showed that the tissues of fish individuals infected with I. multifiliis and the tank water samples were positive detection signals. In contrast, the tissues and water samples from uninfected fish individuals and tanks containing healthy fish showed no signals. The detection results demonstrated the reliability of this detection method. Overall, the novel TaqMan probe-based qPCR method with high sensitivity and specificity as well as repeatability for detection of I. multifiliis was a valuable tool in detecting the parasite in farming water, pond sediments, and fish tissues and could provide early warning for prevention of the disease caused by I. multifiliis.</p>\",\"PeriodicalId\":23716,\"journal\":{\"name\":\"Veterinary parasitology\",\"volume\":\"334 \",\"pages\":\"110381\"},\"PeriodicalIF\":2.0000,\"publicationDate\":\"2024-12-26\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Veterinary parasitology\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://doi.org/10.1016/j.vetpar.2024.110381\",\"RegionNum\":2,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"PARASITOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Veterinary parasitology","FirstCategoryId":"97","ListUrlMain":"https://doi.org/10.1016/j.vetpar.2024.110381","RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"PARASITOLOGY","Score":null,"Total":0}
Establishment and application of TaqMan probe-based quantitative real-time PCR for rapid detection and quantification of Ichthyophthirius multifiliis in farming environments and fish tissues.
Ichthyophthirius multifiliis, a pathogenic ciliate, is a crucial pathogen of freshwater fish and can result in severe economic loss in the aquaculture industry worldwide. It is necessary to develop a sensitive and accurate method for detecting I. multifiliis in farming environments and fish skin and gills to protect fishes from infection of the parasite due to a lack of both safe and effective treatment drugs. The present study established a new TaqMan probe-based quantitative PCR (qPCR) detection method targeting the coding region of the cathepsin L cysteine protease (ICP2) gene of I. multifiliis. The sensitivity, specificity, reproducibility and application for detection and diagnosis of the TaqMan probe-based qPCR method were evaluated. In addition, the linear model between the cycle threshold (Ct) and the logarithmic starting quantity (SQ) of the number of theronts per 1 L of sterile water was developed as Ct = -3.312lg(SQ)+ 34.47 with an R2 of 0.9636 and a minimum detection limit of 4 theronts per 1 L of water and could be employed to determine the theront number based on Ct value. The results of the detection of trial infection samples with the TaqMan probe-based qPCR method showed that the tissues of fish individuals infected with I. multifiliis and the tank water samples were positive detection signals. In contrast, the tissues and water samples from uninfected fish individuals and tanks containing healthy fish showed no signals. The detection results demonstrated the reliability of this detection method. Overall, the novel TaqMan probe-based qPCR method with high sensitivity and specificity as well as repeatability for detection of I. multifiliis was a valuable tool in detecting the parasite in farming water, pond sediments, and fish tissues and could provide early warning for prevention of the disease caused by I. multifiliis.
期刊介绍：
The journal Veterinary Parasitology has an open access mirror journal,Veterinary Parasitology: X, sharing the same aims and scope, editorial team, submission system and rigorous peer review.
This journal is concerned with those aspects of helminthology, protozoology and entomology which are of interest to animal health investigators, veterinary practitioners and others with a special interest in parasitology. Papers of the highest quality dealing with all aspects of disease prevention, pathology, treatment, epidemiology, and control of parasites in all domesticated animals, fall within the scope of the journal. Papers of geographically limited (local) interest which are not of interest to an international audience will not be accepted. Authors who submit papers based on local data will need to indicate why their paper is relevant to a broader readership.
Parasitological studies on laboratory animals fall within the scope of the journal only if they provide a reasonably close model of a disease of domestic animals. Additionally the journal will consider papers relating to wildlife species where they may act as disease reservoirs to domestic animals, or as a zoonotic reservoir. Case studies considered to be unique or of specific interest to the journal, will also be considered on occasions at the Editors'' discretion. Papers dealing exclusively with the taxonomy of parasites do not fall within the scope of the journal.
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号
