Comparison of Quantitative PCR Assays for Detection of

separation-fluorescent immunoassay ... Giardia as the supplemental analysis for the validation of ... Development of quantitative PCR assay specific...

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Comparison of Quantitative PCR Assays for Detection Cryptosporidium Oocysts and Giardia Cysts in Water Samples クリプトスポリジウム等の遺伝子検査法の検討 ○村崎 平林

愛(大阪市水道局) 達也(大阪市水道局)

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武田 万里子(大阪市水道局) 三輪 雅幸(大阪市水道局)

1. Introduction Drinking water works in Japan have monitored regularly for Cryptosporidium oocysts and Giardia cysts in the raw water based on “The Guidelines for Cryptosporidium Treatment in Water” by MHLW. One of the most common methods applied to detect oocysts and cysts from water is microscopy with an immunomagnetic separation-fluorescent immunoassay (IMS-FIA) after concentrating of a large-volume of water sample. Conventional microscopy, however, requires experienced microscopists to accurately identify oocysts/cysts yet results might include a false positive. It is also often difficult to make reassessment or reconfirmation. Based on these facts, the investigative committee for microbiological issues of drinking water, set by MHLW, has surveyed the potential of using PCR technology as a sensitive and specified detection method. The committee examined a quantitative reverse transcription-PCR (RT-qPCR) assay specific for target RNA. The Osaka Municipal Waterworks Bureau (OMWB) has installed a quantitative real-time PCR (qPCR) assay specific for the target DNA of Cryptosporidium/Giardia as the supplemental analysis for the validation of microscopy. This paper will give basic information comparing the characteristics of qPCR and RT-qPCR. Stocked standard oocysts/cysts and necessary reagents for RT-qPCR were offered by the Department of Parasitology of NIID. We will discuss their characteristics according to the results of these sensitive analyses, spike-tests and field surveys. 2. Material and method Ten liters of water were concentrated by the PTFE filtering method. Oocysts/cysts were collected by using sucrose density-gradient centrifugation. The concentrated layer were purified through an immunomagnetic separation (DynabeadsGC-combo;Invitrogen) and examined by microscopy with fluorescent immunoassay (FIA). DNA/RNA of the collected or stocked standard oocysts/cysts were dissolved in TE-buffer solution by five cycles of freezing and thawing (-80 and 56 degrees Celsius) and treated by protein kinase K. Both lysates were centrifuged for 3 min at 12000rpm or 10000G. The supernatants were used for the quantitative real-time PCR (qPCR) and the quantitative reverse transcription-PCR (RT-qPCR) assays. For the qPCR, we used the primers and TaqMan probe and Gene Expression MasterMIx (Takara) whose set for Cryptosporidium was originally designed1) and for Giardia was β –Giardin P2412)3). Both were obtained from a commercial laboratory (Applied Biosystems). For the RT-qPCR, PrimeScript RT Regent Kit and CycleavePCR Detection Kit (Takara) were used. The target gene of Cryptosporidium and Giardia is 18SrRNA. 3. Results and discussion 3.1 Sensitivity analysis The sensitivity of each PCR assay was determined using a known quantity of standard Cryptosporidium oocysts and Giardia cysts which were inactivated by UV processing before being stocked. Standard curves were obtained using DNA extracted from the samples spiked with the equivalent of 3x104-3x10-1 of oocysts/cysts. The curves of RNA were generated with the equivalent of approximately 10-10-4 of oocysts/cysts. While the qPCR can detect the equivalent amount of 0.3 oocysts and cysts, the RT-qPCR can detect the equivalent amount of 0.0006 of oocysts and 0.0012 of cysts. The detection sensitivity of the RT-qPCR was roughly a hundred times higher than that of the qPCR. 3.2 Spike-test Spike-tests on stocked oocysts/cysts from raw water concentrates were carried out. Figure 1 shows the results with comparing the equivalent value between microscopy, the qPCR and the RT-qPCR. The genetic-methods, especially RT-qPCR tended to be higher counts of Cryptosporidium than microscopy. The values of RT-qPCR in particular vary widely during four times of experiments and the quantitative issue will be remained. Relatively similar counts and equivalent numbers of Giardia, however, were obtained between by three methods. The RT-qPCR method would be affected by the bioactivity and the amount of gene expression in an oocyst/cyst individual organism due to use of RNA as a target gene. In addition, since sensitivity is very high, final calculating results are considered to yield remarkable differences. Negative controls were not detected in these experiments, thus their qualitative performances when compared to microscopy were high.

Comparison of Quantitative PCR Assays for Detection Cryptosporidium Oocysts and Giardia Cysts in Water Samples クリプトスポリジウム等の遺伝子検査法の検討

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3.3 Field surveys Microscopy and two quantitative PCR assays were utilized for Cryptosporidium and Giardia in effluents from three waste water treatment plants (WWTPs) in upstream area. The results are shown in Table 1. Cryptosporidium was detected twice by RT-qPCR, but not by the other methods. Detected oocyst genes were confirmed not to be false positive since the sequence analysis on the RT-qPCR products identified the target gene of Cryptosporidium. On the other hand, Giardia was often observed but the counts or the equivalent values varied. Comparing between the results of the simultaneous samples, all values of RT-qPCR were quite smaller than the other methods. The reason for the decreased quantitativity performance of RT-qPCR assay is unclear however a possibility seems to be affected due to degradation of RNA of fragil cysts in the effluents from WWTPs. Considering the results in the spike-test and field surveys, concerning both of the PCR assays, more or less, problems with quantitative performance remained but qualitative results were high. Cryptosporidium

10,000 Oocysts

10,000 Cysts

scope qPCR

1,000

1,000

RT-qPCR

100

100

10

10

Giardia

Fig-1 Result of spike-test

scope qPCR RT-qPCR

1

1 1

2

3

1

4

2

3

(oocysts/cysts in 10L)

Table-1 Result of field survey Cryptosporidium

WWTP A WWTP B WWTP C

Giardia

Scope

qPCR

RT-qPCR

Scope

qPCR

RT-qPCR

1-Sep

ND

ND

ND

4

170

0.005

28-Sep

ND

ND

ND

10

95

0.23

1-Sep

ND

ND

10

26

720

0.035

28-Sep

ND

ND

0.041

ND

ND

ND

6-Sep

ND

ND

ND

10

30

0.14

28-Sep

ND

ND

ND

ND

ND

ND

4. Conclusion Spike-test of the RT-qPCR on Cryptosporidium gave the higher equivalent value, with varying widely. In addition, Cryptosporidium was detected in the effluents from WWTP, despite the negative results by microscopy. This may be due to the high sensitivity of RT-qPCR. We should take into consideration such features more carefully when we deal with a positive result disagreeing with the negative result in microscopy. For spiked Giardia, both experiments on qPCR and RT-qPCR methods revealed acceptably good quantitative performance. For the effluents from WWTP, however, RT-qPCR showed a lack of quantitative performance. Giardia cysts were fragile so that their RNA genes possibly were unstable in contaminated water. According to the results of our investigations, both of the PCR assays give high qualitative performances. However, we, consider that the qPCR is a still better choice for improving detection of Cryptsopridium and Giardia of microscopy as a supplemental examination in OMWB, because it may give more consistent results even for contaminated water. Further improvements of its sensitivity and quantitativity are still future issues. 5. Acknowledgement We are thankful to Dr. Shinji Izumiyama of the Department of Parasitology of the National Institute of Infectious Diseases, who offered standard samples of Cryptosporidium/Giardia and the technique of RT-qPCR. 6. Reference 1)Takeda et. al. 2009. Development of quantitative PCR assay specific for Cryptosporidium and its application. Annual report of Water Examination Laboratory of OMWB. 61:21-25 2)Takeda et. al. 2010. Development of quantitative PCR assay specific for Giardia and its application to environmental water. Annual report of Water Examination Laboratory of OMWB. 62:24-28 3)Rebecca A. Guy et. al. 2003. Real-time PCR for quantification of Giardia and Cryptosporidium in environmental water samples and sewage. Appl. Environ. Micrbiol. 69:51785185