A new method for efficient detection of Cryptosporidium RNA by real-time reverse transcription-PCR with surfactants

Cryptosporidium is one of the most common causes of waterborne diseases worldwide. Its oocysts possess a robust wall that is extremely resistant to the chlorine used for potable water disinfection. The current procedures of nucleic acid extraction and purification, such as the freeze–thaw (F/T) method and the commercial kits, are time consuming and expensive. To this end, a surfactant extraction treatment (SET) was developed as a method to extract nucleic acids from Cryptosporidium using only surfactants. The use of 18S rRNA improves the sensitivity of Cryptosporidium detection for real-time polymerase chain reaction (PCR), because 18S rRNA molecules are constitutively present in high copy numbers. Therefore, we applied SET to the detection of Cryptosporidium 18S rRNA using reverse transcription (RT)-PCR for the first time. RT-PCR was inhibited by 0.01% of the anionic surfactant sodium dodecyl sulfate (SDS), whereas the inhibition did not occur with 5% of the nonionic surfactants Tween 20, Triton X-100, Tween 80, and Triton X-114. However, the nonionic surfactants could not completely suppress the inhibition induced by 0.1% SDS. We successfully extracted 18S rRNA genes from oocysts by SET without the F/T method and detected them by real-time RT-PCR. 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.2015.063 s://iwaponline.com/ws/article-pdf/15/5/1061/414598/ws015051061.pdf Takahiro Sekikawa (corresponding author) Kosuke Toshiki Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, 52–1 Yada, Suruga-ku, Shizuoka 422-8526, Japan E-mail: sekikawa.shizuokakendai@gmail.com


INTRODUCTION
Cryptosporidium is a protozoan parasite distributed worldwide. It is excreted in the feces of infected humans or animals and is the causative agent of cryptosporidiosis, whose symptoms include watery diarrhea, vomiting, and fever. While Cryptosporidium parvum and C. hominis are the primary species known to infect humans, recent studies suggest that C. cervine, C. felis, and C. meleagridis may also cause diarrhea in humans (Carey et al. ). The oocysts of Cryptosporidium spp. are shed in the feces and may then enter sewage treatment facilities via wastewater or persist in the environment. A previous study has demonstrated that some sewage treatments are not efficient enough to eliminate all the oocysts prior to water discharge (Bonadonna et al. ), which can lead to outbreaks of cryptosporidiosis. The largest outbreak of watery diarrhea was recorded in Milwaukee, WI, USA, in 1993 and was caused by Cryptosporidium oocysts that were not removed by the filtration system of one of the city's water treatment plants. Over 400,000 residents in the Milwaukee area presented watery diarrhea, abdominal cramps, fever, and vomiting symptoms (Mac Kenzie et al. ). Cryptosporidium oocysts present a robust wall resistant to several environmental factors as well as to many of the processes and substances normally used for water disinfection.
The robust nature of the oocyst wall requires more stringent treatments for disruption (Carey et al. ). Thus, early detection of oocysts in untreated water sources is essential to ensure efficient quality control for drinking water. Successful detection of nucleic acids from purified oocysts usually requires complex extraction and purification processes aimed at digesting their protective wall. These may involve the use of methods such as freeze-thaw (F/T) cycles, enzymatic treatment, and surfactant treatment prior to nucleic acid amplification. Some of the most common methods used to extract nucleic acids from oocysts include freeze-thawing the samples in a lysis buffer or using commercially available nucleic acid extraction kits with proteinase K and a lysis buffer. The lysis buffer generally contains the anionic surfactant sodium dodecyl sulfate (SDS) (Webster et al. ; Leng et al. ; Nichols & Smith ; Schiffner et al. ). However, SDS is an inhibitor of PCR even at extremely low concentrations; therefore, a cleaning step is required to eliminate any trace of SDS prior to PCR amplification (Weyant et al. ). Furthermore, common procedures to extract and purify nucleic acids, such as the F/T method or those followed using commercial kits, are time consuming and expensive. Thus, there is an interest in the development of faster and more inexpensive methods to extract nucleic acids from oocysts.
In previous studies, we developed a surfactant extraction treatment (SET) as a simple alternative to extract DNA from C. parvum oocysts using only an anionic surfactant for PCR and loop-mediated isothermal amplification (Sekikawa & Kawasaki ; Sekikawa et al. ). Here, we explore the use of nonionic surfactants in suppressing the inhibition induced by SDS and the efficiency of the extraction method in amplifying DNA without a nucleic acid purification step.
The use of 18S rRNA improves the sensitivity of Cryptosporidium detection for real-time PCR, because 18S rRNA molecules are constitutively present in high copy numbers (Fontaine & Guillot ; Kishida et al. ). However, the efficacy of the SET extraction procedure in 18S rRNA detection has not been evaluated. Therefore, we examined the efficacy of SET combined with real-time reverse transcription (RT)-PCR as a fast method for extracting 18S rRNA from C. parvum oocysts.

C. parvum oocysts
Purified and quantified C. parvum oocysts (Iowa isolate) were obtained from Waterborne Inc. (New Orleans, LA, USA). One week after administering the oocysts to C57BL/6 mice orally, the oocysts were purified from fecal samples using a sucrose gradient and Percoll density gradient with centrifugation at

DNA and RNA templates
We extracted and purified nucleic acids by freeze-thawing oocysts between À80 and 37 W C in Tris-EDTA buffer five

RESULTS AND DISCUSSION
Standard curve determination    Table 3 shows the results of the induced inhibition assay. When 0.1% SDS was added to the RT mix, the final concentration in the PCR assay was 0.008%, which could lead to the inhibition of PCR. Therefore, the RT mix was diluted 10 times with TE buffer before PCR to reduce the concentration of SDS below 0.001%. As Table 3 shows, real-time RT-PCR using an RT mix with 0.01% SDS was inhibited and delayed compared with the reaction using RNA templates of dilution ratio of 0.1 corresponding to 2 × 10 À1 oocysts/PCR reaction (Table 2). Furthermore, DNA amplification using an RT mix with 0.1% SDS and a PCR mix with 0.01% SDS was not detected.

Suppression of the inhibition of RT-PCR induced by SDS
To test whether nonionic surfactants can suppress the inhibition of RT and PCR induced by SDS, we examined the effect of combining SDS and nonionic surfactants in the same real-time RT-PCR assay. Table 4 shows the effect of adding nonionic surfactants to RT and PCR assays. DNA amplification was delayed when 0.01% SDS was present in the RT mix, and it was completely inhibited at 0.01% SDS in the PCR mix (Table 3). However, the inhibition induced by 0.01% SDS was suppressed by adding nonionic surfactants to the RT or PCR mix prior to real-time RT-PCR. One microlitre of an RNA template corresponding to 10 oocysts/μl (10 oocysts/RT reaction) was subjected to RT-PCR in the case of the dilution ratio of 1.
n ¼ 2.  The C t value (38.0) in the case of an RT reaction including 0.01% SDS (Table 3) appeared to be increased compared with the C t value (29.1) in the absence of surfactants (positive control) ( Table 2). The C t value (29.6) of DNA amplification using the RT mix containing 0.01% SDS and 5% Tween 20 (Table 4) was almost the same as the C t value (29.1) of the c The RT mix diluted ten times with TE buffer was amplified using PCR.
n ¼ 2. positive control. These results showed that Tween 20 is effective in suppressing the inhibition of RT-PCR induced by SDS.
Test to detect 18S rRNA gene from C. parvum oocysts using SET and real-time RT-PCR Nucleic acids from oocysts were extracted using SET and incubated with or without 5% Tween 20; the RT product obtained was subsequently amplified using PCR (Figure 1(a)). Table 5 shows the results of adding Tween 20 to the RT mix before real-time RT-PCR. One microlitre, 2.5 μl, or 5.0 μl of the SET product was added to each RT mix in a final reaction volume of 10 μl. Thus, the concentrations of the SET products in the RT mixes were 10%, 25%, and 50%, respectively. The maximum volume of the SET product added to the RT mix was 5.0 μl to ensure that the maximum concentration of the sample did not exceed 50% as recommended by the manufacturer. As a result, DNA amplification using the RT products as templates was not detected or was very delayed in the absence of Tween 20. When the RT product included 10% of the SET product and 0.01% SDS in the absence of Tween 20, DNA amplification was not detected. This lack of detection could relate to the low number of oocysts included in the RT reaction (0.08 oocysts) and the inhibition of the RT mix by 0.01% SDS.
When 5% Tween 20 was added to the RT mix, the speed of DNA amplification directly correlated with the concentration of the SET product in the RT mix. The speed of DNA amplification using the 25% SET products in the RT mix (corresponding to 1 oocyst/RT reaction) was faster than that using the RNA template. These results suggested that 5% Tween 20 could suppress the inhibition induced by 0.05% SDS (Table 5). Therefore, the best concentration of the SET product in the RT mix to improve detection sensitivity is 50%. The C t values using an RNA template and using a SET product, both corresponding to 1 oocyst/RT reaction, were 29.1 and 27.0 respectively.
These data prove that SET increases the probability of extracting the 18S rRNA gene compared to F/T and presents the advantage of not requiring a nucleic acid purification step.
Although an autoclave ( However, F/T is known to damage nucleic acids, particularly RNA. These results demonstrate that SET can be successfully used to extract RNA more efficiently than the F/T method.

CONCLUSION
Here we assessed the effectiveness of a new method, SET, for extracting 18S rRNA from purified oocysts. The inhibition of RT-PCR induced by 0.01% SDS could be suppressed by adding one of the four nonionic surfactants tested in this study. Comparing the reactions including 0.1% SDS, a nonionic surfactant, and either RT or PCR products as the template, the C t values observed for Tween 20, Triton X-100, and Tween 80 did not differ greatly.
However, Triton X-114 was particularly weak in suppressing the inhibition induced by SDS. Among the four nonionic surfactants, Tween 20 appeared as the best suppressor of the inhibition induced by SDS. These data show that SET can be used to generate a template for RT-PCR without the need for general nucleic acid extraction methods. Consequently, our results demonstrate