Bottled water testing: RT-PCR, ISO standards, and Nytran™ membranes
Waterborne diseases continue to pose serious global health risks and highlight the importance of access to safe drinking water sources. This blog explores the use of reverse transcriptase polymerase chain reaction (RT-PCR), a highly sensitive method for detecting viruses such as Hepatitis A and norovirus in packaged water products. We also emphasize the role of ISO standards and Nytran™ membranes to enhance virus detection and concentration. These tools and protocols form a robust framework for safe commercially distributed bottled water.
Why should bottled water be tested for viruses?
Despite advances in water and wastewater treatment technology, waterborne diseases pose significant public health challenges worldwide. According to the World Health Organization (WHO) in 2019, unsafe water, sanitation, and hygiene (WASH) services were responsible for 18.3 deaths per 100,000 population (1). This includes deaths from diarrheal diseases, intestinal nematode infections, protein-energy malnutrition, and acute respiratory infections.
The perceived safety of bottled water compared to tap water quality often stems from a lack of trust in local water infrastructure. The bottled water industry utilizes water sources including springs, wells, or municipal supplies and then perform treatment processes that influence the final water quality. While it’s regulated under safety standards, the core driver of increased bottled water consumption in some regions is due to public concern over the reliability and safety of municipal tap water.
Studies show that bottled water is generally free of viral contaminants with no epidemiological link between viral infections and bottled water consumption but is still tested for public health and safety (2). However, there are still risks associated with specific viruses in bottled water (3). In 2016, the Public Health Agency of Catalonia (ASPCAT) reported that 4,136 cases of gastroenteritis were suspected to be linked to bottled spring water from office water coolers, potentially caused by sewage pollution of the spring aquifer(4).
One of the most advanced techniques for detecting viruses in bottled water is reverse transcriptase polymerase chain reaction (RT-PCR). This molecular method amplifies the genetic material of viruses to allow for identification of even trace amounts of microorganisms (5).
For further bottled water safety, it is recommended to use electropositive membranes to concentrate viruses from water samples. These membranes have positively charged surfaces that attract and bind negatively charged virus particles from large volumes of water. In contrast, uncharged membranes do not yield satisfactory results in recovering specific viruses (6). This approach simplifies sample preparation and increases the reliability of virus recovery. By adhering to the guidelines set out in EN ISO 15216-1, reliable and rapid detection of viral pathogens can be achieved (7).
Introduction to ISO 15216-1:2017
The ISO 15216-1:2017 standard outlines a method for the quantitative detection of Hepatitis A virus (HAV) and norovirus genogroups I (GI) and II (GII) RNA in various food samples. These samples include soft fruits, leafy vegetables, stem and onion vegetables, bottled water, bivalve molluscan shellfish (BMS), and food surfaces. For bottled water, the detection process involves adsorption and elution using positively charged membranes followed by concentration through ultrafiltration. This method is applicable to bottled water with a volume up to 2 Liters.
Workflow for ISO 15216-1 for Bottled Water Samples:
- Sampling
- Virus extraction (filtration, elution, concentration)
- RNA extraction
- Real-time RT-PCR
The ISO procedure provides extraction of highly purified RNA, which is essential for accurate detection by RT-PCR. Real-time RT-PCR increases sensitivity and specificity by monitoring the amplification process through measuring the fluorescence of labelled molecules. Due to the complexity of the method, comprehensive controls are necessary for reliable quantification of viral RNA levels in test samples.
Enhancing virus extraction with Cytiva
Cytiva enhances the virus extraction process outlined in ISO15216-1 by offering products that provide precise and reliable virus detection results. Here are some of our offerings to help you achieve these outcomes:
1. Nytran™ membranes
Whatman™ nylon membranes are appropriate for applications involving lower sample loads and are commonly used in Southern and Northern blotting, RNA binding, and RNA sensing. These membranes are available in a highly charged format known as Nytran SuPerCharge (SPC) membrane, which features a very high positive charge. Nytran SPC membranes offer a highly uniform pore size and distribution and provide improved consistency compared to standard nylon membranes. In alignment with ISO 15216-1, an electropositive membrane with a 0.45 μm pore size and 47 mm diameter is recommended. To fulfill these requirements and support efficient virus adsorption, Nytran SPC is our recommended solution. For further details, application guidance, or ordering information, please contact your local Cytiva sales representative.2. Filtration equipment
We offer a variety of vacuum and overpressure filtration systems compatible with electropositive membranes. These systems are available in durable materials such as stainless steel, glass, and high-performance plastic polymers. For laboratories processing a small to medium number of samples, we recommend using a traditional borosilicate glass vacuum filtration system. This setup is known for its chemical resistance and reliability for standard lab workflows.
4013 - Funnel 300 ml with flask 1 L Link to product
For laboratories managing a high volume of daily filtrations, an integrated and efficient solution is essential. The Cytiva stainless steel manifold, when paired with Sentino™ filter funnels, forms a high-throughput filtration system designed for speed, reliability, and ease of use. The manifold provides a durable, easy-to-clean platform ideal for repeated use in demanding lab environments. The Sentino filter funnels are disposable and pre-sterilized to minimize contamination risk and eliminate cleaning and autoclaving steps. Their press-fit design provides secure placement on the manifold for quick setup and membrane handling. This combination streamlines workflows, reduces manual handling for large-scale filtration tasks. When used with Nytran SPC membranes, the system delivers consistent virus adsorption performance in compliance with the ISO 15216-1 standard.
Laboratory manifold
Sentino filter funnels
3. Centrifugal devices
After filtration, the filters are eluted, recovered with an alkaline elution buffer, and further concentrated by ultrafiltration before the final stage. We offer Macrosep™ Advance centrifugal concentrators that are easy to operate and have high concentration efficiency. ISO 15216-1 standards specify requirements for these products, including a capacity of 15 mL and a relative molecular weight limit of 100 kDa. The centrifugal concentrators we recommend for this analysis are:
MAP100C36 - Macrosep Advance centrifugal devices with Omega™ 100K membrane 6 pcs.
MAP100C37 - Macrosep Advance centrifugal units with Omega 100K membrane 24 pcs.
MAP100C38 - Macrosep Advance centrifugal units with Omega 100K membrane 100 pcs.
Macrosep Advance centrifugal devices
Why choose Cytiva?
Our filtration portfolio can be tailored to your requirements, including specific configurations, pore sizes, flow rates, housing sizes, and adapters. With over 250 years of experience in providing high-quality filter products, we will help you find the right solution for your needs. Our worldwide manufacturing facilities are based on quality systems designed to meet the stringent requirements of ISO-9001:2008 and ISO 13485.
Reference:
- World Health Organization. Mortality rate attributed to exposure to unsafe WASH services (per 100 000 population). Published January 8, 24. https://data.who.int/indicators/i/C123B15/ED50112
- Benoît Gassilloud, Gantzer C. Adhesion-Aggregation and Inactivation of Poliovirus 1 in Groundwater Stored in a Hydrophobic Container. Applied and Environmental Microbiology. 2005;71(2):912-920. doi:10.1128/aem.71.2.912-920.2005
- Zhao Z, Hossain MI, Jung S, et al. Survival of murine norovirus and hepatitis A virus in bottled drinking water, strawberries, and oysters. Food Control. 2021;133:108623. doi:10.1016/j.foodcont.2021.108623
- Blanco A, Guix S, Fuster N, et al. Norovirus in Bottled Water Associated with Gastroenteritis Outbreak, Spain, 2016. Emerging Infectious Diseases. 2017;23(9):1531-1534. doi:10.3201/eid2309.161489
- Hennechart-Collette C, Dehan O, Laurentie M, Fraisse A, Martin-Latil S, Perelle S. Method for detecting norovirus, hepatitis A and hepatitis E viruses in tap and bottled drinking water. International Journal of Food Microbiology. 2022;377:109757. doi:10.1016/j.ijfoodmicro.2022.109757
- Hennechart-Collette C, Dehan O, Fraisse A, Martin-Latil S, Perelle S. Evaluation of three different filters and two methods for recovering viruses from drinking water. Journal of Virological Methods. 2020;284:113939. doi:10.1016/j.jviromet.2020.113939
- International Organization for Standardization. ISO 15216-1:2017 Microbiology of the food chain — Horizontal method for determination of hepatitis A virus and norovirus using real-time RT-PCR — Part 1: Method for quantification. https://www.iso.org/standard/65681.html
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