4.1. Materials and Methods
4.1.1. Evaluation of NoV reduction yield in electropositive filters
As described in Fig. 4.1, NoV reduction yield was evaluated by measuring human NoV absorption degree of each electropositive filter, respectively.
4.1.1.1. Test sample
After spiking 2.5 X 105 copies of human NoV which was isolated from patient’s stool sample, it was put into 50 liters of sterilized distilled water to produce test sample.
4.1.1.2. Electropositive filter
1MDS filter (3M, St. Paul, MN, USA), 3M filter (not for sale, 3M) and Nanoceram filter (Argonide, Sanford, FL, USA) were selected as the electropositive filters for this study.
4.1.1.3. Quantification of human NoV using Real-time RT-PCR
Water processed with the 1-MDS filter produced a final elute of 20 ml.
Viral RNA was extracted from 140 μl of that elute using the QIAamp® Viral
protocol to obtain the final volume of 60 μl. Real-time PCR was conducted using a Bio-Rad iScript One-step RT-PCR Kit (Hercules, CA, USA). To quantify the NoV, real-time RT-PCR amplifications were performed with primer sets (Table 4.1) and real-time RT-PCR conditions (Table 4.2).
Twenty microliter of viral RNA as the template and 30μl of the premixed kit solution were mixed for the reaction. The real-time RT-PCR was carried out using MyiQ real-time PCR machine (Bio-Rad), according to the following protocol.
Figure 4.1. Procedure for quantification of human NoV.
Table 4.1. Primer and probe of Human NoV used in Real-time RT-PCR
Primer & probe Sequence (5’→3’)
Cog2F (GII) CAR GAR BCN ATG TTY AGR TGG ATG AG
Cog2R (GII) TCG ACG CCA TCT TCA TTC ACA
Ring2 (GII) FAM-TGG GAG GGC GAT CGC AAT CT-BHQ
Table 4.2. Real-time RT-PCR conditions
Reaction Conditions
Reverse transcription
50℃ 10 min 1 cycle
Initial denaturation
95℃ 5 min
1 cycle 95℃ 10 sec
Amplification 55℃ 30 sec 45 cycle
4.1.2. Evaluation of NoV reduction yield in combined water treatment system
As described in Figure 4.2, NoV reduction yield was evaluated by measuring human NoV absorption and elimination degree in combined water treatment system.
4.1.2.1. Test sample
After spiking, 8.0 X 106 copies (1st trial) and 9.1 X 106 copies (2nd trial) of human NoV which was isolated from patient’s stool sample and itwas dispersed into 50 liters of sterilized distilled water to produce test sample.
4.1.2.2. Combined water treatment system
Combined water treatment system was composed of chlorine feeder, UV irradiation device and electropositive filter cartridge (Figure 4.2) and overall water treatment system consists of filtration using 3M filter, UV irradiation (39 W/m2)and chlorine disinfection (0.5mg/L) (Figure 4.3). Virus spiking sample was passed through the combined water treatment system
Figure 4.2. Combined water treatment system.
Figure 4.3. Composition and flow diagram of combined water treatment system.
Water In
3M filter cartridge
UV irradiation
Chlorine disinfection
Water out
20 L / min
5 inch electropositive filter
39 W / m2
0.5 mg / L
Figure 4.4. Overall evaluation procedure of reduction yield of NoV after water treatment using combined water treatment system.
Combined system
Adsorption to 1 MDS filter
Elution
Concentration
RNA extraction
Real-time RT-PCR Human NoV spiking
Control
4.1.2.3. Quantification of human NoV using Real-time RT-PCR
Water processed with the 3M filter produced a final eluate of 20 ml.
Viral RNA was extracted from 140 μl of that elute using the QIAamp® Viral RNA Mini kit (Qiagen) according to the manufacturer’s protocol to obtain the final volume of 60 μl. Real-time PCR was conducted using a Bio-Rad iScript One-step RT-PCR Kit.
To quantify the NoV, Real-time RT-PCR amplification was performed with primer sets (Table 4.1) and Real-time RT-PCR conditions (Table 4.2).
We used 20μl of viral RNA as the template and 30μlof the premixed kit solution. The real-time RT-PCR was carried out using MyiQreal-time PCR machine (Bio-Rad), according to the following protocol.
4.2. Results and Discussion
4.2.1. Evaluation of NoV reduction yield in electropositive filters
Electropositive filters are effective to capture the majority of virus particles from the water passing through the filter and several electropositive filters of various compositions are now commercially available. Real-time RT-PCR result of human NoV, described in Figure 4.5, shows that these electropositive filters work efficiently for removal of NoVs. From the filter test twice for statistical analysis, the average human NoV reduction yields of 3M filter, 1MDS filter and Nanoceram filter were 42.05%, 33.85 % and 35.30%, respectively (Table 4.3). And these two tests of each electropositive filter showed consistent result for reduction of NoV in water and very low standard deviations (especially for 3M filter), suggesting their high fidelity for NoV removal. Capture capacity of human NoV on the 3M filter was equivalent or better than those of others. Therefore, 3M filter was used for further water treatment and evaluation of this combined water treatment system.
Table 4.3. Human NoV reduction yield of electropositive filters
Filter type 1st trial (%) 2nd trial (%) Average
Standard deviation
1-MDS 32.0 35.7 33.85 2.616
3M 42.2 41.9 42.05 0.212
Nanoceram 34.0 36.6 35.30 1.838
4.2.2. Evaluation of NoV reduction yield in combined water treatment system.
Due to high fidelity of 3M filter for reduction of human NoVs in water, 3M filter was selected and used for development of combined water treatment system. This system consists of 3M filter cartridge, UV irradiator and chlorine feeder. To evaluate this system, the human NoV reduction yield was tested. For quantification of human NoV particles, quantitative real-time RT-PCR was conducted. To test this, 6.9 X 105 copies of human NoV particles in the spiking sample were quantified using this quantitative real-time RT-PCR system. The RT-PCR result showed that quantified amount of the spiking sample is very close to the original amount, suggesting that this method is very useful for quantification of human NoVs in water (Figure 4.6). To evaluate this combined water treatment system, two spiking samples, 6.9 X 105 and 7.9 X 105 copies of human NoV particles, were prepared (Table 4.4). These two spiking samples were passed by the treatment system and amount of human NoV particles in eluted samples after water treatment
by this system. Therefore, this combined water treatment system can be very useful for water treatment to significantly reduce more than 5 logs of human NoV particles from the groundwater.
108 107 106
105
104
103 102 101 Spiking
Table 4.4. Human NoV reduction yield of combined water treatment system
Control Combined system*
1st trial 6.9 X 105 copies ND
2nd trial 7.9 X 105 copies ND
* , ND, not detected.
4.3. Conclusion
To develop the novel combined water treatment system using electropositive filtration, UV irradiation, and chlorine disinfection, three commercially available electropositive filters were tested and 3M electropositive filter was selected, due to high fidelity of human NoV reduction in water. This filter can reduce more than 40% of human NoVs in water, confirmed by quantitative real-time RT-PCR. After selection of electropositive filter, the combined water system was developed and evaluated using spiking samples containing more than 105 copies of human NoV particles. After treatment of the spiking samples, no human NoV was detected in both eluted samples from the system, suggesting that this combined water treatment system can reduce more than 105 copies of human NoV particles or can remove human NoV particles completely. Therefore, this system should be applied for groundwater treatment in schools and industries to make sure the safety of groundwater for drinking.
PART V: References
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초 록
양전하 필터와 자외선 살균기, 염소소독기를 복합적으로 이용하는
복합수처리장치를 제작하였다. 105 copies 이상의 노로바이러스를
식수에 spiking 하여 시료를 만들고 복합수처리장치를 이용한 처리구에 대해 real-time RT-PCR 을 이용하여 정량분석하였다.
그 결과 물에 존재하는 노로바이러스에 대하여 105 이상의
제거효과가 있음을 확인하였고 지하수의 노로바이러스 오염에 대한 해결책으로써 복합수처리장치가 효과적으로 적용될 수 있음을 확인하였다.
주요어 : 노로바이러스 Sequence, Phylogenetic tree, 지하수, 복합수처리장치, 양전하필터, 노로바이러스 제거
학번 : 2005-31092