Detection method and kit for cryptosporidium in drinking water

Abstract

The invention discloses a detection method and a kit for cryptosporidium in drinking water. The method comprises the following steps: a disc filter is used for retaining microorganisms in the drinkingwater by filtering, a filter membrane is dissolved by acetone, the microorganisms are precipitated by phosphate, freeze thawing pretreatment is performed, external structures of oocysts are broken byprotease, a supernatant is obtained through centrifugation and used for directly extracting oocyst DNA, and qPCR based gene quantification is performed. When the cryptosporidium in the drinking wateris detected with the detection method, false positive is avoided, results are accurate and reliable, the specificity is high, 10 SSUrRNA gene copies can be detected, the sensitivity is high, the detection results are obtained fast, experimental cost is low, and recovery rates, of cryptosporidium in deionized water and an underground water source, determined with the detection method reach 55% and46% respectively.

Description

technical field [0001] The invention relates to the technical field of environmental monitoring and microbial molecular detection, in particular to a detection method and kit for cryptosporidium in drinking water. Background technique [0002] Cryptosporidium disease caused by Cryptosporidium can be transmitted through drinking water, which poses a major threat to human health, especially for people with low immunity, which can cause fatal diarrhea. Cryptosporidium sclerenchyma oocysts with a diameter of 4-6 μm can resist hypochlorous acid disinfection in conventional water treatment and survive in water for several months, and are still infectious after 13 months in 4°C water [Chen Fu, 2008. Animals Research on molecular epidemiology, pathogenic mechanism and anti-infective drugs of Cryptosporidium [D]. Doctoral dissertation of Nanjing Agricultural University.] The detection of Cryptosporidium in drinking water can prevent the epidemic and occurrence of diseases and ensure ...

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Problems solved by technology

At present, the detection of Cryptosporidium in domestic and international drinking water is mainly based on the separation of immunomagnetic beads and fluorescence microscopy [Cui Yanmei, Wang Yunxia, ​​Duan Xiaohui, et al. Research progress[J]. Journal of Food Safety and Quality Inspection, 7(4):1369-1374; Yu Suhua, Chen Yiqiu, Yan Jinling. 2009. Determination of Giardia cysts and Cryptosporidium oocysts in water by immunofluorescence method[J]. Environmental Monitoring Management and Technology, 21(1):33-35; KingD.N., Donohue M.J., Vesper S.J., et al.2016. Microbial pathogens in source and treated waters from drinking water treatment plants in the United States and implications for human health [J].Science of the Total Environment,562:987-995.], this is also the standard method for China's GB / T5750 national standard and the U.S. EPA Cryptosporidium detection, the method uses immunomagnetic separation reagents to separate oocysts, and uses immunomagnetic separation Fluorescence technology is used for dyeing microscopy, and microscopic observation is more dependent on human subjective judgment. Some fluorescent antibodies have high background signal and low specificity, which may easily lead to inaccurate results [Lemos, V., Graczyk, T., Alves, M .,et al.2005.Identification and determination of theviability of Giardia lambila cysts and Cryptosporidium parvum andCryptosporidium hominis oocysts in human fecal and water supply samples byfluorescent in situ hybridization(FISH)and monoclonal antibodies[J].Parasitolog,48-Research 53; Quintero-Betancourt W. 2003 Assessment of Current and New Methods for Investigating the Occurrence of Enteric Pathogens in Ambient Waters and Water Sources for Reh ydrationof a FloridaWetland.University of South Florida,2003.], and immunomagnetic separation reagents are expensive, which limits the wide application of this method

[0003] The development of molecular biology has promoted the application of molecular technology in the detection and diagnosis of pathogens. In recent years, molecular methods are being developed to detect Cryptosporidium. Molecular methods have fast detection speed and high sensitivity. Positive rate or qPCR gene quantification [Gu Youfang, Wang Kai, Liu Deyi, et al. 2015. Molecular detection of Giardia lamblia and Cryptosporidium infection in pet dogs [J]. (33) 5:362-367; Chalmers R M and KatzerF.2013.Looking for Cryptosporidium:the application of advances in detection and diagnosis[J].Trends in Parasitology,29(5):237-251;Chalmers R.M and KatzerF.2013.Looking for Cryptosporidium:the application of advances in detection and diagnosis[J].Trends in Parasitology,29(5).], but currently there is no mature and acceptable method for molecular detection of Cryptosporidium in drinking water or source water, and there are still some problems in sample pretreatment. The problem

When the collected sample is relatively low (less than 2.5L), it is usually filtered with a filter membrane of about 47mm, and the DNA of the sample is directly extracted by cutting the filter membrane, and then quantitatively analyzed for Cryptosporidium genes [Guy, R.A., P.Payment, U.J. Krull, and P.A. Horgen. 2003. Real-time PCR for Quantitation of Giardia and Cryptosporidium in environmental water samples and sewage [J]. Applied and Environmental Microbiology, 69: 5178–5185; Wang Mingxing, Bai Yaohui, Liang Jinsong, etc. .2016. Quantitative detection of common pathogens in water by FCM-qPCR method[J].Environmental Science, 26(1):384-390.], the sample volume collected is low, so the DNA content is correspondingly reduced, resulting in detection reduced range, does not correctly indicate Cryptosporidium contamination

When the amount of sample collected is large (more than 5L), most of the current reports still rely on the method of immunomagnetic separation to separate oocysts [Almeida A, Moreira M, Soares S, Delgado M, Figueiredo J, Silva E, Castro A Costa A and CostaJ.2010.Biological and Genetic Characterization of Cryptosporidium spp.AndGiardia duodenalis isolates from five hydrographical basins in Northern Portugal.Korean J Parasitol.,48(2):105-111; Helmi K,Skraber S,Burnet J,LeblancL,Hoffmann L and Cauchie H. 2011. Two-year monitoring of Cryptosporidium parvumand Giardia lamblia occurrence in a recreational and drinking water reservoir using standard microscopy and molecular biology techniques. Environ MonitAssess, 179:163–175; Xiao L. Alderisio K. and Ji 2006.Detection of Cryptosporidiumo oocysts in water:effect of the number of samples and analytic replicates on test results[J].Applied and environmental microbiology,72(9):5942–5947.], high experimental cost is not conducive to wide application

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