A new immunomagnetic bead separation – surfactant extraction treatment protocol for rapid and sensitive quantitative PCR detection of Cryptosporidium parvum DNA

The Cryptosporidium oocyst is encased in a robust wall that is extremely resistant to detrimental environmental factors such as chlorine used to disinfect potable water. Therefore, extracting oocyst DNA is not a trivial undertaking. Standard procedures used to extract DNA from oocysts, such as freeze–thaw (F/T) methods and DNA purification kits, are time-consuming and expensive and are difficult to implement in routine clinical practice. Therefore, we developed a surfactant extraction treatment (SET) that efficiently extracts DNA from the oocyst. Immunomagnetic separation (IMS) combined with quantitative real-time polymerase chain reaction (qPCR) detects pathogenic microorganisms with high sensitivity. The objective of the present study was to evaluate SET for its ability to simplify qPCR detection of 18S rDNA directly from immunomagnetic bead–oocyst conjugates. DNA extracted directly from the conjugates using SET did not affect DNA amplification in the qPCR assay. Further, the rate of DNA amplification using IMS–SET was greater than that using F/T combined with the DNA purification kit. The rate of recovery of oocysts from surface water samples spiked with oocysts did not differ significantly from previously published values. These data demonstrate that the new IMS–SET protocol using qPCR can simplify the recovery and detection of Cryptosporidium oocysts. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying, adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/). doi: 10.2166/ws.2016.125 s://iwaponline.com/ws/article-pdf/17/1/161/410646/ws017010161.pdf Takahiro Sekikawa Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 4228526, Japan E-mail: sekikawa.shizuokakendai@gmail.com

the largest outbreak associated with a contaminated public water source reported to date in the United States, Cryptosporidium made 403,000 residents of Milwaukee, WI sick (Corso et al. ). This outbreak was caused by Cryptosporidium oocysts that were not removed by one of the city's water treatment plants, and the victims experienced watery diarrhea, abdominal cramps, fever, and vomiting (MacKenzie et al. ). In 2010, a waterborne cryptosporidiosis outbreak occurred in Östersund, Sweden, and affected at least 12,700 inhabitants (Widerström et al. ).
The infectious dose varies depending on the Cryptosporidium isolate, and as few as 10 oocysts cause disease (Okhuysen et al. ). Cryptosporidium represents a major challenge to the delivery of safe drinking water because the oocysts can survive and remain infectious for 16 months in disinfectant-treated water supplies (Bridle et al. ). The oocysts are encased in a durable oocyst wall comprising inner and outer walls composed of a protein-lipid-carbohydrate matrix that serves as a highly effective protective barrier against deleterious environmental factors (Fayer ). Thus, early detection of oocysts in water sources is essential to ensure the safety of drinking water (Widerström et al. ).
Successful detection of nucleic acids from purified oocysts usually requires complex extraction and purification processes aimed at digesting their protective wall. These protocols may involve techniques such as freeze-thaw (F/T) cycles as well as treatments using enzymes and surfactants before nucleic acid amplification. These procedures take at least 1 h (Gao et al. ).
Recently, we developed a new methoda surfactant extraction treatment (SET) that is simple to perform and inexpensive (Sekikawa & Toshiki ). SET uses an anionic surfactant, sodium dodecyl sulfate, or SDS, a common component of lysis buffers generally (Webster et  A cycle threshold analysis tool integrated in the LightCycler Nano software V1.1.0 was used to compute threshold levels.

Effect of IMBs subjected to SET on qPCR assays
The effects of 2 μL of IMBs (Dynabeads anti-Cryptosporidium [Invitrogen Dynal AS, Oslo, Norway]) with or without SET on a qPCR assay were determined using a DNA standard (3 × 10 4 copies/μL). The IMBs occupied 10% of the volume of the master mix.
Each reagent was independently prepared in a PCR tube. The PCR for each experiment was run together using the LightCycler Nano system (Roche Diagnostics).

Detection of Cryptosporidium DNA in IMB-oocyst conjugates subjected to SET
The effect of SET, and of IMB-oocyst conjugates subjected to SET, on qPCR assays was determined using standard suspensions of 10 5 oocysts added to 10 mL distilled water in a 10-mL centrifuge tube. IMB-oocysts were prepared and isolated using 100 μL of the IMB kit described above,

Standard curve
Cryptosporidium parvum oocysts were used to generate a standard curve. Figure 2 shows the amplification of dilutions of the DNA extracted from the oocysts using SET. Ct values were calculated for 2 to 2 × 10 4 oocysts per PCR assay. Each point on the line represents the mean of three independent PCR amplifications. The slope ¼ À1.447, and the correlation coefficient (r 2 ) ¼ 0.9979.

Effect of IMBs subjected to SET on qPCR assays
The results of qPCR assays using the DNA standard with or without IMBs are shown in Table 1. In the absence of Tween 20, the Ct values of the untreated IMBs and the positive control were 23.09 and 23.14, respectively, indicating that a 10% suspension of IMBs did not inhibit the qPCR assay.
DNA isolated from the IMBs after SET without Tween 20 was not amplified (Table 1). In our previous study, the final concentration of SDS in the PCR mix was 0.01%, Figure 2 | Standard curve generated from 2 to 2 × 10 4 oocysts of C. parvum per PCR assay (n ¼ 3, r 2 ¼ 0.9979). DNA was extracted from the oocysts using SET. DNA detection from river water samples spiked with oocysts that were subjected to IMS-SET We tried detecting Cryptosporidium DNA in the water sample obtained from the Okitsu River using filtration,

IMS, F/T and the DNA purification kit (DNeasy Blood &
Tissue kits; QIAGEN). PCR inhibitors were removed from the river water sample by using the DNA purification kit.
As a result, Cryptosporidium DNA was not detected on the qPCR assay. Therefore, the Okitsu River sample was used for the preparation of a river water sample spiked with oocysts.
Oocysts were recovered from samples of distilled water or river water using IMS, and DNA was extracted from IMB-oocyst conjugates using SET. The limit of detection of the qPCR assay was two oocysts/PCR reaction mixture (Table 3) Tween 20 using a 10 À1 dilution was not detected because the concentration of SDS was 0.001%.
In general, oocyst recoveries depend on the compositions of water samples because river water contains inhibitory turbid materials (Fontaine & Gulliot ).
Here, when we determined the ability of IMS-SET without Tween 20 to detect Cryptosporidium DNA in the river water sample diluted to 10 À1 concentration (0.001% SDS), DNA amplification was not detected (Table 3). There are various substances and particles in the river water sample. Some substances and particles attach to the surface of IMB-oocyst conjugates. The SET product contains 0.1% SDS, free oocyst DNA and the conjugates with the substances and particles.
Serial dilutions of the SET product (1-10 4 fold) were used to prepare the samples for PCR assays. Therefore, there is a possibility that the substances or the particles affected DNA amplification. DNA in the river water sample diluted to 10 À2 concentration was able to be amplified as well as DNA in the distilled water sample because PCR inhibitors The result of the recovery rates of Cryptosporidium oocysts from water samples spiked with 10 5 oocysts is shown in Figure 3. The recovery rates of IMS-SET from 10 mL-distilled water (DW), centrifuge-SET from 10 mL-DW, centrifuge-F/T-DNA purification from 10 mL-DW, IMS-SET from 10 L-DW and IMS-SET from 10 L-river water were 100.0 ± 2.0% (positive control), 103.5 ± 4.0%, 77.0 ± 9.0%, 80.0 ± 9.0% and 60.8 ± 7.8%, respectively. Of the three 10 mL-DW samples, the recovery rate of only the centrifuge-F/T-DNA purification sample was decreased. These results indicate that the process of F/T or DNA purification was not able to recover the Cryptosporidium DNA from the oocyst as well as SET.
Substances in the river water sample were not analyzed in the present study. However, the presence of large feed particles in raw water interferes with the ability of IMS to recover oocysts, and certain substances and particles inhibit PCR assays (Fontaine & Guillot ).  qPCR, quantitative real-time PCR; SE, standard error; IMS, immunomagnetic separation; SET, surfactant extraction treatment; ND, none detected. a 0.01% SDS. n ¼ 3. IMS was used to recover oocysts from 10 L of DW or river water spiked with 10 5 oocysts. F/T, freeze-thaw. n ¼ 3. Error bars express standard errors of the mean. IMS or centrifugation was used to collect oocysts from DW or river water samples spiked with 10 5 oocysts. Oocyst DNA was extracted using SET or F/T-DNA purification. Each water sample without dilution was added to a PCR tube. The concentration of Tween 20 in a PCR tube was 5%. The recovery rates of oocyst from samples were calculated using a standard curve.

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T. Sekikawa | A new protocol for Cryptosporidium DNA detection Water Science & Technology: Water Supply | 17.1 | 2017