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Monitoring infectious disease threats in environmental water sources

Infectious waterborne diseases, which can be transmitted by drinking water that is contaminated by exposure to soil, sewage, and infected animals, pose a serious threat to the safety of the water supply. Global climate change increases the risk of outbreaks because higher temperatures can promote the growth of pathogens and the vectors that transmit them and increase the likelihood of exposure to contaminated water sources.

Leptospirosis is a waterborne disease caused by the Leptospira bacteria that can provoke kidney damage, meningitis, liver failure, respiratory distress, and even death. It infects humans through exposure to the urine of animals carrying these bacteria or environmental samples contaminated with urine from an infected animal. While leptospirosis has a worldwide distribution, it particularly affects countries with tropical and subtropical climates (Taniguchi and Póvoa 2019). This disease spreads easily during climate catastrophes, and extreme weather is a high-risk factor, as in the case of the leptospirosis outbreak in Puerto Rico following Hurricane Maria in 2017.

An effective method for monitoring the presence of Leptospira bacteria in the environment is crucial for preventing and controlling leptospirosis outbreaks. Since this disease can be transmitted by infected animals that contaminate water supplies, it is necessary to analyze samples from a variety of animal as well as water sources in order to identify the source of infection. In the following study, researchers harnessed the power of multiplex PCR to develop an assay that can accurately distinguish Leptospira bacteria from other species and is sufficiently robust to provide accurate results from environmental samples containing PCR-inhibitory contaminants.


Tracking the source of infection

A research group in Okinawa, Japan (Sato et al. 2019) sought to develop new tools to systematically detect Leptospira in order to prevent human infection. This group studied the bacterial ecosystem that allows the development of Leptospira during biofilm formation and investigated which animals are potential reservoirs for transmitting these pathogens to humans.

The researchers screened environmental water samples from a known endemic region in Japan for rRNA targets specific to Leptospira and animals living nearby. They performed multiplex PCR analysis with Takara Ex Taq HS DNA polymerase to detect bacteria using 16S rRNA targets, which they analyzed by NGS (on an Illumina MiSeq® platform). A similar procedure was carried out using PrimeSTAR HS DNA polymerase to detect 12S rRNA from vertebrate animals in the same environmental samples, in order to understand which vertebrate species are more likely to harbor Leptospira (Figure 1). The presence of certain animals, especially boars and eels, seemed to correlate with high levels of Leptospira, showing a potential link between pathogen and carrier. They were able to draw a correlation between the main bacteria (12 strains) that help propagate pathogenic Leptospira and the animals (10 species) that are the primary reservoirs of these bacteria. The multiplex PCR method used in the study is a powerful tool to help determine how Leptospira outbreaks can occur by showing how the environment can impact the development of this pathogen and revealing how it interacts with hosts/carriers. In addition to providing a better understanding of this phenomenon, these findings helped develop a system to better predict human infection risk.

Library preparation procedure for metabarcoding sequencing based on a two-step tailed PCR

Figure 1. Schematic view of the library preparation procedure for metabarcoding sequencing based on a two-step tailed PCR. Multiplex PCR was applied in the first step for Leptospira and bacterial detection. The procedure for vertebrate mitochondrial 12S rRNA sequencing was basically the same but slightly modified from that of Miya et al. 2015. Image and caption adapted from Sato et al. 2019 and used under a Creative Commons Attribution 4.0 International License.


Why our polymerases are the investigative tools of choice

This study highlights the use of Takara Ex Taq HS and PrimeSTAR HS DNA polymerases to detect bacteria in environmental water samples, which are typically difficult to work with due to the presence of PCR-inhibitory contaminants. It demonstrated the robustness of these two enzymes for performing DNA amplification with samples which pose such challenges. Since screening assays can help prevent health disasters provoked by weather crises, which are occurring with greater frequency and severity due to climate change, having reliable reagents to power those assays will be crucial for getting ahead of future outbreaks.


References

  • High Prevalence of Deadly Bacterial Disease Found in Puerto Rico. Yale School of Medicine. Available at: https://medicine.yale.edu/news-article/20887/
  • Leptospirosis | CDC. Available at: https://www.cdc.gov/leptospirosis/index.html
  • Miya, M. et al. MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: Detection of more than 230 subtropical marine species. R. Soc. Open Sci. 2, (2015). Available at: https://pubmed.ncbi.nlm.nih.gov/26587265/
  • Sato, Y. et al. Environmental DNA metabarcoding to detect pathogenic Leptospira and associated organisms in leptospirosis-endemic areas of Japan. Sci. Rep. 9, 1–11 (2019). Available at: https://www.nature.com/articles/s41598-019-42978-1
  • Taniguchi, L. U. & Póvoa, P. Leptospirosis: one of the forgotten diseases. Intensive Care Med. 45, 1,816–1,818 (2019). Available at: https://link.springer.com/article/10.1007/s00134-019-05839-z

Featured products

Cat. # Product Size License Quantity Details
RR006A TaKaRa Ex Taq® DNA Polymerase Hot-Start Version 250 Units USD $257.00

A hot-start version of Takara Ex Taq DNA polymerase, which combines the proven performance of Takara Taq polymerase with the proofreading activity of an efficient 3'-to-5' exonuclease, for high-sensitivity, high-efficiency PCR. Ex Taq is optimized for amplicons up to 20 kb from genomic DNA, and up to 30 kb from lambda DNA. Separate tubes of Mg2+ plus buffer and dNTP mix are supplied with the hot-start polymerase.

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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The amplification efficiencies of Takara Ex Taq HS DNA Polymerase and a high-grade hot start PCR enzyme from Company A were compared

The amplification efficiencies of Takara Ex Taq HS DNA Polymerase and a high-grade hot start PCR enzyme from Company A were compared

The amplification efficiencies of Takara Ex Taq HS DNA Polymerase and a high-grade hot start PCR enzyme from Company A were compared. A 7.5 kb target was amplified from increasing amounts of human genomic DNA template. Excellent sensitivity and yields were obtained with Takara Ex Taq HS enzyme. Lane M: Lambda-Hind III digest, Lane 1: 100 pg, Lane 2: 300 pg, Lane 3: 1 ng, Lane 4: 3 ng, Lane 5: 10 ng, Lane 6: 30 ng, Lane 7: 100 ng.

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RR006A: TaKaRa Ex Taq DNA Polymerase Hot-Start Version

RR006A: TaKaRa Ex Taq DNA Polymerase Hot-Start Version
R010A PrimeSTAR® HS DNA Polymerase 250 Units USD $233.00

License Statement

ID Number  
M54 This product is covered by the claims of U.S. Patent Nos. 7,704,713 and its foreign counterparts. 

A high-fidelity hot-start (HS) PCR DNA polymerase with superior proofreading ability due to robust 3' to 5' exonuclease activity. PrimeSTAR HS DNA polymerase can efficiently amplify up to 8.5 kb for human genomic DNA targets or up to 22 kb for lambda DNA.  Separate tubes of optimzed buffer (Mg2+ plus) and dNTP mix are supplied with the enzyme.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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PrimeSTAR High Amplification Efficiency

 PrimeSTAR High Amplification Efficiency
PrimeSTAR High Amplification Efficiency. Amplification efficiency was compared using high fidelity enzymes from Company A and Company B. (Target: Human DCLRE1A gene [2kb]) Reaction mixtures were prepared and PCR cycling conditions were performed according to each company's protocol (50 µL PCR reaction). These results demonstrate that PrimeSTAR provides excellent amplification efficiency with higher specificity than other suppliers' high fidelity enzymes. In addition, the detection sensitivity was higher by one order of magnitude.

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Sequence analysis of amplified DNA fragments

Sequence analysis of amplified DNA fragments
Sequence analysis of amplified DNA fragments. Sequence analysis is the most accurate method to investigate mutation frequency. This analysis revealed that PrimeSTAR HS has higher fidelity than an alternative high fidelity enzyme from Company A, with only 12 mismatched bases per 249,941 total bases, and 10X higher fidelity than Taq DNA polymerase. These results demonstrate PrimeSTAR HS DNA Polymerase's suitability for PCR amplifications that require extreme accuracy.

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R010A: PrimeSTAR HS DNA Polymerase

R010A: PrimeSTAR HS DNA Polymerase

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