WO2011043737A1

WO2011043737A1 - Viability analysis of protozoa using polymerase chain reaction (pcr)

Info

Publication number: WO2011043737A1
Application number: PCT/SG2010/000379
Authority: WO
Inventors: Haiqing Gong; Chao Ping Lou
Original assignee: Nanyang Technological University
Priority date: 2009-10-05
Filing date: 2010-10-05
Publication date: 2011-04-14

Abstract

The present invention relates to a method for detecting viable protozoa cells in a sample fluid comprising the steps of: (i) contacting a sample fluid with a phenanthridium compound capable of preferentially penetrating dead or membrane-compromised cells over viable and/or substantially intact cells to intercalate with at least one nucleic acid molecule to; (ii) exposing the sample fluid to a light source to substantially covalently bind nucleic acid molecules of dead or membrane-compromised cells to the phenanthridium compound to form a reacted sample; (iii) separating the cells from any excess phenanthridium compound; (iv) lysing the viable and/or substantially intact cells to release nucleic acid molecules into solution; and (v) performing a PCR on the nucleic acid solution with PCR primers specific to the protozoa, wherein detection of amplification indicates the presence of viable and/or substantially intact protozoa cells.

Description

Viability analysis of protozoa using polymerase chain reaction (PCR) Field of the invention

The present invention relates to the detection of organisms, for example, in environmental sampling, food and water safety, pathogen detection and disease control. The invention in particular relates to the detection and/or differentiation of viable cells, for example protozoa.

Background of the invention

The detection of viable or live organisms presents an important challenge for many applications. For example, in the case of pathogenic microorganisms, it is important to identify viable or live cells as these cells are metabolically active and/or reproductive with the potential to infect and cause diseases. In particular, the ability to determine the concentration of viable cells is important.

For example, detection of viable Cryptosporidium is very crucial for water monitoring. Cryptosporidium was once an emerging pathogen but now it is an established human infectious waterborne and food borne pathogen. A major outbreak in 1993 in Wisconsin, Milwaukee, USA affected an estimated 403,000 people (Kaminski et al., 984). In the event of such outbreaks it may be vital to determine the concentration of viable Cryptosporidium oocysts present in the drinking water sample in order to make accurate risk estimations. Current methods of Cryptosporidium contamination of water networks are done by USEPA method 1622 and method 1623. These methods are based on microscopy staining of concentrated samples but do not provide any information on the viability of the oocysts.

However, traditional microscopy methods alone are not able to detect viable and/or live organisms, such as protozoa, for example Cryptosporidium parvum. In recent years, the methods adopted for testing viability of Cryptosporidium parvum oocysts in water samples are largely based on staining with fluorogenic dyes like PI/DAPI with microscopic observation, in-vitro excystation, cell culturing and animal infectivity methods.

With the fluorogenic dyes (DAPI & PI) method, propidium iodide (PI) and 4', 6- diamidino-2-phenylindole, dihydrochloride (DAPI) are fluorescent dyes used in the most commonly adopted method for assessment of survival of C. parvum oocysts under various environmental pressures. The blue-fluorescent DAPI nucleic acid stain preferentially stains DNA (Gasser et a/., 1999). PI is also a fluorescent dye which can penetrate into the dead cells walls with compromised membrane permeability. Sporozoite nuclei, which take up DAPI but fail to stain with PI, are viable (i.e. DAPf , ΡΓ) and termed ghost cells or empty cells, while cells that stain with both fluorochromes are nonviable (i.e. DAPI^", ΡΓ). This method requires skilled labour, a fluorescence microscope and is labour intensive and a lengthy procedure (i.e. time consuming). Moreover, this dye permeability assay was found to be unreliable and was not able to indicate the viability of the oocysts during oocysts disinfection studies (Korich er a/., 1990).

In the in vitro excystation method to determine oocysts viability, the host body environment is simulated, and this includes creating a variety of conditions like addition of bile salts, pancreatic juices and setting up appropriate environmental conditions. After excystation, the oocysts are microscopically observed for released or partly released sporozoites. However, this method is effective only for high concentrations of oocysts, and is not feasible for application to testing of environmental water samples where the oocysts concentration is very low.

With mammalian cell culturing, cultured cell lines are infected by oocysts samples and observed for parasite antigen after 24-48 hours by Foci Detection method. The success of mammalian cell culturing depends upon various factors like the cell lines used, the purity of oocysts suspension, pre-incubation excystation treatment and centrifugation techniques of the sample (Carey et a/., 2004). Moreover, this technique also requires specialized equipment and experienced personnel to handle and maintain cultures, which are not applicable for routine testing of water samples.

Animal infectivity methods are considered the most direct method to investigate : the viability of oocysts. Samples are directly injected into live mice, followed by observation of diarrheal symptoms and shedding of oocysts in faeces. Peeters et al., 1989 found that 1000 oocysts per mouse were required to induce 100% infection and therefore the method is not easily adaptable to routine environmental testing. Moreover, animal testing is very expensive and time consuming.

In Cell Culture-PCR, seeded oocysts are concentrated by filtration and centrifugation, and inoculated onto a human colon carcinoma (Caco-2) cell line (Rochelle et al., 1997). Intensive labour is required for the growth and maintenance of the cell monolayer and the prolonged incubation period represent significant disadvantages of the cell culture technique, particularly in confirming a potential waterbome outbreak where time is of essence. However, the technique remains a good substitute for animal infectivity models.

It is desirable to develop efficient methods of differentiating viable cells.

Summary of the invention The present invention relates to a method for detecting viable cells. This method importantly concentrates, differentiates, separates and detects the viable cells. According to a first exemplary aspect, the method for detecting viable protozoa in a sample fluid comprises the steps of: i) contacting a sample fluid with a phenanthridium compound capable of preferentially penetrating dead or membrane-compromised cells over viable and/or substantially intact cells to intercalate with at least one nucleic acid molecule; (ii) exposing the sample fluid to a light source to substantially covalently bind nucleic acid molecules of dead or membrane-compromised cells to the phenanthridium compound to form a reacted sample;

(iii) separating the cells from any excess phenanthridium compound;

(iv) lysing the viable and/or substantially intact cells to release nucleic acid molecules into solution; and

(v) performing a PCR on the nucleic acid solution with PCR primers specific to the protozoa, wherein detection of amplification indicates the presence of viable and/or substantially intact protozoa cells. Any suitable method for separating the cells from excess phenanthridium compound may be used. In particular, the cells may be separated by using a filter and then recovered from the filter for further analysis.

Accordingly, in a second exemplary aspect, there is provided a method for detecting viable protozoa in a sample fluid comprising the steps of: (i) contacting a sample fluid with a phenanthridium compound capable of preferentially penetrating dead or membrane-compromised cells over viable and/or substantially intact cells to intercalate with at least one nucleic acid molecule;

(ii) exposing the sample fluid to a light source to substantially covalently bind nucleic acid molecules of dead or membrane-compromised cells to the phenanthridium compound;

(iii) separating the cells of the sample from any excess phenanthridium compound using a filter;

(iv) recovering the cells from the filter; (v) lysing the viable and/or substantially intact cells to release the nucleic acid molecules into solution; and

(vi) performing a PCR on the nucleic acid solution with PCR primers specific to the protozoa, wherein detection of amplification indicates the presence of viable and/or substantially intact protozoa cells.

Brief description of the figures

The invention will be better understood with reference to the drawings, in which:

Figure 1 depicts an example of a filter apparatus.

Figure 2 depicts a device for exposing a tube to a blue LED light. Figure 3(a) depicts a real-time PCR reaction for quantifying DNA to determine the extraction efficiency and 3(b) depicts the real-time standard curve for C. parvum for the logarihmic amplification plots for 10² , 5 x 10³, 10⁴ and 10⁶ oocysts per tube (x-axis: no. of cells, y-axis: Crossing threshold, Ct value).

Figure 4 depicts a real-time PCR result showing the effect of PMA on amplification of DNA from inactive C. parvum cells. Plot 1 is for inactive C. parvum without PMA treatment with a Ct value of 26.99; Plot 2 is for inactive C. parvum treated with 12.5 μΜ PMA with a Ct value of 32.1 1 ; Plot 3 is for inactive C. parvum treated with 25 μΜ PMA; Plot 4 is for C. parvum treated with 50 μΜ PMA.: ^' ;: . Figure 5 depicts a real-time PCR result showing the effect of PMA on amplification of DNA from viable C. parvum cells. Plot 1 is for viable C. parvum without PMA treatment, with a Ct value of 24.95; Plot 2 is for inactive C. parvum treated with 12.5 μΜ PMA with a Ct value of 25.04; Plot 3 is for inactive C. parvum treated with 25 μΜ PMA with a Ct value of 26.17; Plot 4 is for C. parvum treated with 50 μΜ PMA with a Ct value of 26.43. Figure 6 depicts a real-time PCR result showing the amplification from 5000 viable C. parvum oocysts without PMA treatment and a mixture of 5000 viable and 5000 inactive C. parvum oocysts with 25 μΜ PMA. The amplification from 5000 viable C. parvum gave a Ct value of 25.02 and the amplifcation from the mixture gave a Ct value of 25.30,

Figure 7 ; shows the agarose gel electrophoresis of the real-time PCR of Figure 6. Lane 1 : 100 bp DNA ladder; Lane 2: DNA extraction from 5000 viable C. parvum oocysts without PMA treatment; Lane 3: DNA extraction from a mixture of 5000 viable and 5000 inactive C. parvum oocysts with 25 μΜ PMA. Detailed description of the invention

The present invention relates to a method to detect viable protozoa in a sample fluid. The sample fluid is contacted with a phenanthridium compound capable of preferentially penetrating dead or membrane-compromised cells over viable and/or substantially intact cells to intercalate with at least one nucleic acid molecule.

The phenanthridium compound may be any phenanthridium compound capable of preferentially penetrating dead or memberane-compromised cells over viable and/or substantially intact cells. After penetrating the dead or membrane- compromised cells, the phenanthridium compound intercalates with at least one nucleic acid molecule and covalently binds with the nucleic acid molecule on exposure to a light source. In particular, the compound propidium monoazide (PMA) may be used. The light source may be a light emitting diode (LED). In particular, the light source may emit blue light to enable the reaction to occur. Covalently bound nucleic acid molecules from dead and/or membrane- compromised cells will not amplify during PCR. After exposure to the light source, the viable cells are separated from any excess phenanthridium compound using a filter. The viable cells are then recovered from the filter.

Any filter may be used to separate the viable cells from excess phenanthridium compound. The filter may comprise a filter membrane with pores of smaller size than the target protozoa to be detected. For example, for Cryptosporidium which are in the range of 3-6 μητι, a suitable pore size may be ~2 μηη.

Any conventional filter may be used, for example, including but not limited to cellulose ester filters, polycarbonate filters and the like. In particular, filter membranes with a uniform pore size may be used. An example of a filter membrane with uniform pore size which may be used is described in WO 2010/110739, the entire contents of which is herein incorporated by reference.

The filter membrane may be contained in a filter apparatus for the filtration. An example of a conventional filter apparatus that may be used for filtration is illustrated in Figure 1. The filter apparatus comprises a lid 1 with an inlet 2, a membrane filter 3 on a filter support 4 and an outlet 5. Another example of a filter apparatus suitable for the invention is described in WO 2010/110739.

After filtration, the trapped protozoa are recovered from the filter membrane and collected for further analysis. Recovery of cells from the filter membrane may be by any method. For example, the membrane filter may be immersed in a solution with agitation (e.g. tangential or lateral shaking) to dislodge the cells into the solution, and subsequently using the solution comprising the dislodged cells for further manipulation and/or analysis. As an alternative example, cells may be recovered from the membrane filter by flushing the cells from the filter membrane, for example as described in WO 2010/ 10739. Separation of the viable cells from excess phenanthridium compound is performed before cell lysis. This separation step reduces the possibility of nucleic acid molecules released from the lysed cells binding to excess phenanthridium compound and not amplifying in the PCR. This separation step may also separate the viable cells from the covalently bound nucleic acid molecules of dead or membrane-compromised cells, which may inhibit or interfere with the PCR. Accordingly, only nucleic acid molecules from viable cells are amplified in the subsequent PCR step.

Lysis of the cells to release nucleic acid molecules into solution may be performed by any method, including but not limited to chemical lysis (for example using guanidine thiocyanate, proteinase K and/or any other chemical suitable for lysis), mechanical lysis, ultrasound lysis, thermolyis, freeze-thawing and electroporation. The cells are lysed to extract the nucleic acid molecules from within the cells. Following lysis, the nucleic acid may be used directly for PCR analysis. Alternatively, the nucleic acid molecules may be extracted and/or further purified before performing PCR analysis.

PCR analysis is performed with PCR primers specific to the target protozoa. Only nucleic acid molecules from the viable cells which were lysed are amplified during the PCR since the covalently bound nucleic acid molecules from dead and/or membrane-compromised cells will not be , amplified. Accordingly, detection of amplification in the PCR indicates the presence of viable protozoa.

PCR analysis may be conventional PCR analysis with detection of amplification may be through agarose gel electrophoresis. Alternatively, the PCR analysis may be real-time PCR as this provides quantitative analysis. The method of the invention may be carried out in a fluidic device and thus may be automated. The method of the invention is suitable for detecting viable protozoa from any fluid sample, including but not limited to environmental, clinical and water samples. The fluid sample may be in liquid form. If the sample is not in liquid form, the sample may be resuspended in a suitable liquid. In particular, the method of the invention is for detecting viable protozoa, for example pathogenic protozoa in water samples, for example, reservoir and drinking water samples. Non-limiting species of viable pathogenic protozoa that may be detected include Crytosporidium (e.g. Cry^ptosporidium parvum), Giardia (e.g. Giardia duodenalis), Entamoeba (e.g. Entamoeba histolytica). Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention.

EXAMPLES

Materials and methods Viable and inactive C. parvum oocysts (bovine, Iowa isolate) were purchased from Waterborne Inc. (New Orleans, LA, PSA). The purified oocysts suspension was supplied in de-ionized water and stored at 4 °C until use. According to the manufacturer, the oocysts were inactivated by heat-killing at 72 °C water bath for 15 minutes. Optimization of PMA treatment

PMA (Biotium Inc., USA) was added at final concentrations of 12.5 μΜ, 25 μΜ or 50 μΜ to sample tubes containing 1000 viable or heat-inactivated C. parvum oocysts in 50 μΙ. Samples were incubated in the dark for 5 min to allow penetration of PMA into dead or membrane-compromised cells. Samples were then exposed to a 20 W blue LED light for 5 min using the device depicted in Figure 2. The device includes a heat cooling fan 1 and heat sink 2 to cool the device when in operation. The tube 4 is placed beneath the stand 3 as shown in Figure 2. When the device is switched on, the tube 4 is exposed to the blue light from a LED beneath the heat sink 2.

Control samples used in the study were 50 μΙ of viable or inactivated C. parvum oocysts not exposed to PMA but incubated in the dark for 5 minutes and exposed to the blue LED light for minutes. All samples and control were further concentrated by evaporation to 5 μΙ.

DNA isolation and quantification

C. parvum oocysts DNA was extracted using a freeze-thaw method: 5 consecutive cycles of freezing in liquid nitrogen for 1 min and thawing at 65°C for 1 min. 1 % Triton X-100 were used as lysis agent. Samples were treated with proteinase K (200 pg/ml) at 56°C for 30 min. DNA was precipitated using isopropanol method. The DNA pellets were resuspended in 50 μΙ TE. 2.5 μΙ were used in PCR, the rest of the DNA was quantified using the Quant-iT picoGreen dsDNA reagent (Invitrogen Inc., USA) according to the manufacturer's instructions.

Quantitative PCR

The DNA sequence (GeneBank accession number XM_626 22, SEQ ID NO: 3) uniquely present in Cryptosporidium species was used as the target for relative quantification of DNA extracted from C. parvum oocysts. Real-time quantitative PCR was performed in a total volume of 10 μΙ containing 1 μΙ extracted genomic DNA and final concentrations of 1 * PCR buffer, 3 mM MgCI₂, 1 * SYBR Green, 0.2μ of forward primer (5'- C AAAC AAGG AGG AATCAG-3 ' ; SEQ ID NO: 1), 0.2μΜ of reverse primer (5'- CTTCATAATCCGGCTAAA-3; SEQ ID NO: 2) using a Rotor-Gene real-time PCR machine (Qiagen, Germany). The cycling parameters were: 2 min at 95 °C followed by 40 cycles of 5 sec at 95 °C, 5 sec at 55 °C and 5 sec at 72 °C. All the PCR products were verified by gel electrophoresis.

Result:

DNA extraction and standard curve DNA extraction efficiency from viable C. parvum oocysts was evaluated using Quant-iT picogreen dsDNA reagent (Invitrogen Inc., USA) and real-time PCR. Quant-iT picogreen dsDNA reagent was used according to the manufacturer's instructions to quantitate the amount of DNA extracted from viable oocysts. The amount of DNA was converted to number of oocysts based on a theoretical content of 195 fg of DNA per C. parvum oocyst. Each sample was then analysed by real-time PCR.

For example, the cycle threshold value was determined from each real-time PCR curve in Figure 3(a). This was plotted against the number of oocysts calculated based on the theoretical DNA content formula to give a standard curve illustrated in Figure 3(b). The measured number of oocysts of the samples was compared to the known number of oocysts used initially. The combined picogreen and real-time PCR analysis measured the DNA extraction efficiency at 93.6 %.

Inactivated C. parvum oocysts PMA as added to inactivated C. parvum oocysts at concentrations of 12.5 μΜ, 25 μΜ and 50 μΜ followed by incubation and LED light exposure as describe above. DNA was isolated by a combination of lysis and freeze-thaw cycles treatment and quantified by real-time PCR. It can be seen from Figure 4 that the real-time PCR generated from the inactive C. parvum oocysts was totally removed by PMA treatment at concentrations of 25 μΜ and 50 μΜ. Viable oocysts

Viable oocysts suspensions were treated with PMA at concentrations of 12.5 μΜ, 25 μΜ and 50 μΜ followed by incubation and LED light exposure as describe above. Real-time PCR amplification was detected at all concentrations of PMA tested including the control (Figure 5). It was observed that PMA at 25 and 50 μΜ may have acted as a potential PCR inhibitor as slight inhibitions of the PCRs were observed (Figure 5).

Mixture of viable and inactive C. parvum oocysts

PMA was added to a mixture of 5000 viable C. parvum oocysts and 5000 inactive C. parvum oocysts at a final concentration of 25 μΜ. Following incubation, LED exposure and DNA extraction described above, real-time PCR analysis was performed. A control sample with 5000 viable oocysts not exposed to PMA as described above was also analysed by real-time PCR. It can be seen from Figure 6 that the viable oocysts could be differentiated in the mixed samples. The similarity in the real-time PCR curves of the control and the mixed sample suggests that DNA from inactivated C. parvum oocysts in the mixed sample did not amplify nor affect the PCR reaction. The PCR product was verified by gel electrophoresis as shown in Figure 6.

Conclusion PMA is a chemically modified version of propidium iodide (PI) with an azide group added to the phenanthridine ring allowing chemical cross-linkage to organic molecules upon short exposure to blue light. Photo-induced cross- linkage disables the DNA from being amplified. This study shows that PMA does not penetrate membranes of live C. parvum oocysts, whereas it is efficiently taken up by dead C. parvum oocysts with permeabilized cell membrane. In combination with rapid real-time PCR, the detection method can be completed within 20 mins. This study suggests the potential of- this PMA- real-time PCR method to be developed as a rapid and sensitive method in monitoring viable C. parvum contamination levels in the drinking water supply system.

References Carey et al., (2004). "Biology, persistence and detection of Cryptosporidium parvum and Cryptosporidium hominis oocyst." Water Research 38(4): 818-862.

Gasser, E. b. R. B. and P. O'Donoghue (1999). "Isolation, propagation and characterisation of Cryptosporidium." international Journal for Parasitology 29(9): 1379-1413. Kaminski ef a/., (1994). "Cryptosporidium and the Public Water Supply." N Engl J Med 331 (22): 1529-1530.

Korich et al. (1990). "Effects of ozone, chlorine dioxide, chlorine, and monochloramine on Cryptosporidium parvum oocyst viability." Appl. Environ. Microbiol. 56(5): 1423-1428. Peeters, J. E., E. A. Mazas, et al. (1989). "Effect of disinfection of drinking water with ozone or chlorine dioxide on survival of Cryptosporidium parvum oocysts." Appl.. Environ. Microbiol. 55(6): 1519-1522.

Rochelle, P., D. Ferguson, et al. (1997). "An assay combining cell culture with reverse transcriptase PCR to detect and determine the infectivity of waterborne Cryptosporidium parvum." Appl. Environ. Microbiol. 63(5): 2029-2037.

WO 2010/110739

Claims

1. A method for detecting viable protozoa in a sample fluid comprising the steps of:

(i) contacting the sample fluid with a phenanthridium compound capable of preferentially penetrating dead or membrane-compromised cells over viable and/or substantially intact cells to intercalate with at least one nucleic acid molecule;

(iv) recovering the cells from the filter;

(v) lysing the viable and/or substantially intact cells to release the nucleic acid molecules into solution; and

(vi) performing a PCR on the nucleic acid solution with PCR primers specific to the protozoa, wherein detection of PCR amplification indicates the presence of viable and/or substantially intact protozoa cells.

2. The method according to claim 2, wherein the filter comprises a filter apparatus comprising a filter membrane.

3. The method according to any one of the preceding claims, wherein the filter membrane has uniformly sized pores. The method according to claim 2 or 3, wherein recovering the cells from the filter comprises agitating the filter membrane to dislodge the cells into a solution or flushing the cells from the filter membrane.

5. The method according to any one of the preceding claims, wherein lysing the cells is by chemical lysis, mechanical lysis, ultrasound lysis, thermolyis, freeze-thawing or electroporation. 6. The method according to any one of the preceding claims, wherein the nucleic acid molecules are purified before PCR.

The method according to any one of the preceding claims, wherein the phenanthridium compound comprises propidium monoazide.

8. The method according to any one of the preceding claims, wherein the light source comprises blue light.

The method according to any one of the preceding claims, wherein the PCR comprises real-time PCR.

The method according to any one of the preceding claims, wherein the protozoa comprises Cryptosporidium, Giardia and Entamoeba species. 1 1. The method according to any one of the preceding claims, wherein the protozoa comprises Cryptosporidium parvum, Giardia Entamoeba histolytica.

12. The method according to claim 11, wherein the protozoa comprises Cryptosporidiun parvum and the PCR primers comprises SEQ ID NO: 1 and SEQ ID NO: 2.

13. The method according to any one of the preceding claims, wherein the sample solution comprises a water sample.