Cryptosporidiosis A Gastroenteric Disease Biology Essay

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Cryptosporidiosis is a gastroenteric disease which results from an infection with the protozoan parasite cryptosporidium parvum . The primary route of infection in humans in the western developed world is via water-borne transmission. Recent decades has witnessed a seemingly exponential demand for clean water supplies thus there is the constant need for vigilance to ensure drinking water supplies are water-borne pathogen free. Predictably however, the number of water-borne infection instances for C.parvum has risen, with this pathogen becoming far more prevalent in recent years.

With entry into the molecular era and the subsequent movement towards the post-molecular era Polymerase Chain Reaction (PCR) has replaced the immune-florescence assay (IFA) as the method of choice for detection of C.parvum oocysts in water samples.

This investigation aimed to utilize a novel Invitrogen© iPrep™ ChargeSwitch® Forensic kit as a means to increase the sensitivity of a Nested-PCR for the detection of DNA isolated from oocysts of Crytptosporidium parvum at very low concentration. It aimed to demonstrate the capability of this procedure to reproducibly detect oocyst at concentrations of less than or equal to 2.5 oocysts per 50µl. In order to achieve this aim; three sequential objectives had to be achieved.

Initially to establish the nested-PCR assay was operational within normal parameters and to create an internal positive control (IPC), a nested-PCR was carried out on a C.parvum DNA sample which had been previously extracted from oocysts by the established P.A.L.M laser micro-dissection (LCM) DNA extraction technique.

Secondly having achieved a positive amplification product using the P.A.L.M laser micro-dissection (LCM) DNA extraction technique, a novel Invitrogen© iPrep™ ChargeSwitch® Forensic kit was employed. It represented a new method of C.parvum DNA extraction from oocysts. A nested-PCR was performed which would confirm or reject the validity of this Invitrogen© kit as an alternative means of DNA extraction applicable to nested-PCR. This conclusion would be achieved by drawing a direct comparison between the amplification products of the two DNA extraction techniques with the IPC representing the DNA extracted by the established P.A.L.M LCM technique.

Finally have shown the Invitrogen© iPrep™ ChargeSwitch® Forensic kit was applicable to use with the nested-PCR assay, a series dilution of C.parvum DNA extracted via the Invitrogen© kit would allow a sensitivity detection limit to be established for a nested-PCR which utilized this novel Invitrogen© iPrep™ ChargeSwitch® Forensic kit.

Thus the intrinsic value inherent to this research correlates to a precise evaluation of the sensitivity detection limit of a nested-PCR assay after the introduction a novel method of nucleic-acid extraction.

Acknowledgements

I would like to thank my project supervisor Dr.Colm Lowery for his time and expertise throughout this project. I also wish to acknowledge my family for their continued support and understanding.

Declaration

I hereby declare that all the body work present is this study which is being put forward to achieve a Masters in Biomedical Science is indeed the work of Mr. Cliff Gilligan unless stated otherwise.

Signature

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Abbreviations

Mobile Genetic Elements: (MGE's)

Lateral Gene Transfer: (LGT)

Horizontal Gene Transfer: (HGT)

Pathogenicity Islands: (PAI)

1.0 Introduction

Cryptosporidiosis

Cryptosporidiosis is the disease associated with Cryptosporidium. Cryptosporidium are protozoan parasites which pertain to the phylum Apicomplexa. Cryptosporidium are regarded as intracellular, obligate parasites which pertain and infect the lower part of the intestinal tract thereby causing diarrhea in a plethora of vertebrate hosts including birds and animals (Morgan and Upton 2000; Monis and Saint 2000). Immuno-compromised patients specifically AID's afflicted individuals will suffer severe and often fatal symptoms relating to this disease.

Routes of Transmission

The innate ability of Cryptosporidium to infect a myriad of animals and because of the ubiquitous presence of cryptosporidium oocysts within the environment, numerous routes of transmission exist for humans to acquire cryptosporidium infection. These include direct contact with an infected individual (person to person transmission infection) or via animals (zoonotic transmission infection), ingestion of contaminated food (food borne infection transmission) or drinking infected water (water borne infection transmission) (Fayer and Xiao 2008).

Cryptosporidium

To date twenty valid species and greater than 40 genotypes of Cryptosporidium have been identified thus far (Chalmers and Davis 2009). Xiao and Feng in 2005 cited five cryptosporidium species/genotypes as being responsible for the majority of human cryptosporidium cases which include C.parvum, C.meleagridiz, C.felis and C.canis (Xiao and Feng 2005;.

C.parvum and C.hominis are responsible for an estimated 90% of reported cryptosporidium cases in the western world with the remaining percentage attributed to C.meleagridis, C.canis or C.felis (Fayer and Xiao 2008).

Cryptosporidium parvum (C.parvum)

C.parvum is considered to be the most prevalent water-borne pathogen in the western world. Transmission infection into individuals is caused by ingestion of sporulated oocysts by the fecal-oral route. C.parvum has displayed resistance to all safe to-human levels of water chlorination and has displayed a survival ability of up to 24 hours plus in 1000 mg per liter of free chlorine. It has also displayed an innate resistance to bleach based disinfectants. The symptoms associated with this infection in an individual are acute and include (dilute) watery and non-blood containing diarrhea. In immune-compromised individuals such as AID's suffers diarrhea excretion levels can reach 10-15 Liters per day (Fayer and Xiao 2008))

Cryptosporidium hominis (C.hominis)

C.hominis and C.parvum share many comparable characteristics including indistinguishable oocyst morphology and life cycle systems. C.hominis is considered an intracellular, obligate parasite that infects the gastrointestinal tract which causes gastroenteritis upon colonization resulting in severe diarrhea. It has also displayed a board resistance to chlorine and bleach based disinfectants and thus is extremely prevalent in water supplies in the developed western world (Zhou et al., 2002).

However in contrast to C.parvum, which can be deemed to have a board host infection range, C.hominis is nearly entirely a human parasite. It has a somewhat low zoonotic potential when a comparison is drawn to C.parvum. Its primary route of transmission into infected individuals is via the fecal-oral route through ingestion by drinking water contaminated with oocysts laden desecration (Virginia Commonwealth University 2008).

Pathogenicity

A pathogen is an infectious biological agent that causes disease or illness to its host. The term is primarily used for agents that disrupt the normal physiology of a multi-cellular animal or plant.

Pathogen- organism with a demonstrated capacity to cause disease

Virulence- relative degree of pathogenicity

The factors by which viruses achieve pathogenicity are referred to as the virulence factors; hence virulence is a measure of a pathogens disease causing capacity (Nicklin et al 2002).

Cryptosporidium entry into the host organism is accommodated for the most part by the environment although geographical location directly influenced the route of transmaission into humans. In the western world water has been recognised as a primary reservoir for the transmission of human enteropathogens such as cryptosporidium (Balkis, A 2010).

The study of virulence/antiobiotic resistance genes will require discussion of homologous genes and proteins. Homology among genes or proteins reflects evolution by divergence from a common ancestor. When two homologous genes in different species have the same function, they are known as orthologs; when two genes in the same or different species have different functions they are known as paralogues. (Nicklin et al 2002).

Conventional Treatment

Treatment of C.parvum was extremely limited until quite recently and centred on supportive therapy such as IV fluids. Both Paromomycin and Nitazoxanide have recently been FDA approved and are implemented to alleviate the diarrheal symptoms. However the effectiveness of Nitazoxanide in immune-compromised patients such as AID's suffers is uncertain and is thus viewed as contraindicated for such groups (Virgina commonwealth University CSBC, 2008).

Antiretroviral drugs are therefore employed in such instances to boost the immune system and manage infection (Rossignol, J.F. 2008).

Cryptosporidium Life-Cycle

An asexual stage and a sexual stage are integrated into the life cycle of cryptosporidium parvum. Post ingestion, the oocysts exist within the small intestinal tract. They release sporozoites that form an attachment to the microvilli of the epithelial cells within the small intestines. Sporozoites after attachment become trophozoites that reproduce asexually by multiple fission in a process known as schizogony. The trophozoites develop into type I meronts that contain 8 daughter cells and type II meronts which contain 4 type II merozoites (http://www.cdc.gov; Ryan et al., 2004). The merozites in question after being released form an attachment to the brush border of the epithelial cells where they diversify into (female) macrogamonts and (male) microgamonts (Chen et al., 2003). Microgamonts pertaining to macrogamonts form zygotes. Zygotes develop into two morphofunctional distinct forms of oocysts. Thick walled oocysts are excreted into the environment whereby they can survive within various environmental conditions for months.

Fig 1.0 Cryptosporidium life-cycle (Fayer and Xiao 2008)

File:Cryptosporidiosis 01.png

Oocysts

As the apicomplexans are exclusively parasitic, an independent oocyst forms a distinctive aspect of the C.parvum life-cycle which is referred to as the spore phase. Upon entry into the host C.parvum oocyst will undergo sexual replication via merogony (schizogony) or sporogony (Zhou et al., 2002).

Cryptosporidium parvum genome

The NCBI in recent times completed the sequencing of the genome of C.parvum and published the findings under the work entitled "Complete genome sequence of the apicomplexan, Cryptosporidium parvum" (Abrahamsen et al., 2004). The genome of C.parvum is now understood to consist of a relatively small and simple organisational structure of 9.1MB. It consists of eight chromosomes respectively, ranging from 1.04 to 1.5Mb in size. The genome of C.parvum is very densely structured and is now shown to contain no transposable elements. C.parvum also displays no genes present in its plastids or mitochondria (Abrahamsen et al., 2004)

Conserved Domains

Conservation relates to change which has occurred at specific positions on amino acid sequences via reassortment. Thus conserved domains are the functional modules of proteins that remain invariable (unchanged) despite various other reassortment changes which may have occurred on that protein sequence.

A domain is a discrete portion of a protein assumed to fold independently of the rest of the protein due its essential function.

1.6.2 Pathogenicity islands

Pathogenicity islands (PAI's) are regarded as a distinct class of genomic islands acquired primarily via later or horizontal gene transfer, which are incorporated within the genome of the pathogenic micro-organism. Designated as occupying relatively large genomic regions from 10-200kb they encode specific genes correlating or orchestrating virulence. PAI's may be deemed as discrete genetic units flanked by direct repeats, insertion sequences or tRNA genes which are sites for recombination into the DNA (Shnakar et al., 2002).

Pertaining to the sequenced C.parvum, Abrahamsen and his colleagues investigation highlighted very few possible PAI's and thus heavily skewed interest toward an immunodominant ≥900kDa protein referred to as GP900 (Abrahamsen et al., 2004)

Pathogenicity Genes

The sequencing of the genome of C.parvum has directed investigation research toward a confirmed and several putative surface proteins thought to enhance pathogenesis, in particular a ≥900kDa protein which is immunodominant referred to as GP900.

This protein has been localized to the apical end of sporozoites and micronemers of merozoites. Post translation glycosylation has been the suggested reason for its high molecular mass and the structure of GP900 displays similarities to that of a family of glycoproteins referred to as mucins.

Barnes et al., believe the GP900 mediates attachment and invasion of host cells. It has also been suggested the protein in question dictates a role in C.parvums resistance to proteolysis by the myriad of proteases located in the mammalian gut (Barnes et al., 1998).

Conventional Detection immune-fluorescence assay (IFA)

The standard conventional technique employed for the routine detection of cryptosporidium spp, in water centers around the immune-fluorescence assay (IFA). This entails the use of labeled cryptosporidium-specific antibodies and fluorescence microscopy (e.g USEPA method, 1623, USEPA, 1999) (Monis and Saint 2000). The microscopy aspect integrates the use of the vital vital staining dyes 4,'6-diamidino-2-phenylindole, dihydrochoride (DAPI) and propidium iodine (PI) to label DNA. This procedure essentially pertains to two classes of immune-fluorescence techniques; primary and secondary

Primary- single antibody chemically linked to a flurophore.

Secondary- two antibodies utilized, the first (the primary antibody) recognises the target molecule and binds. The second (the secondary antibody) which carries the flurophore recognises the primary antibody and binds to it (Monis and Saint 2000).

Nested Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR) is a molecular technique adept at amplifying DNA through a temperature-mediated process. This application requires primers which are complementary to the termini of the target DNA. The amplification products can be utilized in downstream sequencing or analysis along with application uses in DNA fingerprinting within the field of forensic science (Laxer, M.A 1991).

The nested-PCR technique is a modification of the conventional PCR technique. It integrates two sets of primers (F1; R1 and F2; R2) into a sequential operational procedure- it is envisaged to reduce sample contamination in amplification products as the secondary primer set amplifies the secondary target. This secondary target is located within the first run PCR amplification product. This ensures it is extremely improbable that the amplification product from the second round of PCR has contamination from undesired products of primer dimers; hairpins or alternative primer target sequences (Fayer and Morgan 2000; Leng et al., 1996).

Nucleic Acid extraction techniques

1.10.1 P.A.L.M Laser Capture Micro-dissection (LCM)

The P.A.L.M Laser Capture Micro-dissection (LCM) tool is relatively novel and a highly innovative technique utilized for dissecting out (isolation) of viable cells of interest or small cell populations and extracting DNA from such isolates for molecular (functional genomics and proteomic) analysis (Petrovic et al., 2004).

The laser technology is centred around the fact the UV wavelength of the laser pulsed beam is too long to harm polynucleotides and proteins. It is therefore able to excise viable cells or populations of cells from slide-mounted tissue. The isolated tissue is then recovered via a laser capapult technique into a sterile microfuge tube cap. Sunnotel and his collegues are credited with the combination of LCM and Real-time PCR to detect cryptosporidium spp. From tissue mounted onto a glass slide (Hendolin et al., 2006).

Fig 2.0 Schematic diagram of Laser capture micro-dissection (LCM) method (Hewddlin et al., 2006).

1.10.2 Invitrogen© iPrep™ ChargeSwitch® Forensic kit

The Invitrogen© iPrep™ ChargeSwitch® Forensic kit details a novel method for the rapid and efficient purification of genomic DNA from forensic samples. In this instance, the isolation of C.parvum DNA from oocysts. This technique integrates magnetic bead-based technology for the effective isolation of the genomic DNA; sample preparation is conducted via a simple lysis priocedure with Proteinase K which ensures minimal contamination of the sample of interest. The purified genomic DNA will exhibit enhanced downstream performance in applications such as PCR as contaminants and PCR inhibitors have been isolated and washed away in the removal process.

Fig 3.0 stepwise approach to purify genomic DNA from forensic sample using The Invitrogen© kit (Invitrogen© iPrep™ ChargeSwitch® Forensic kit)

Lyse Sample

↓ Bead charge

Bind DNA to ChargeSwitch®

Magnetic beads

Charge on pH ≤6.0

↓

Wash beads containing DNA to remove contaminates

Charge on pH= 7.0

↓

Elute DNA from Beads

Charge off pH= 8.5

1.10.2.1 Invitrogen© Magnetic Micro-bead Technology

The Invitrogen© iPrep™ ChargeSwitch® magnetic-beads facilitates a switchable surface charge dependent on the PH of the integrated buffer which allows nucleic acid purification. This application is centred around a positive charge that binds the negatively charged nucleic acid backbone, Proteins and other such sample contaminants are not bound. Raising the pH to 8.5 elutes the nucleic acids as the innate charge located on the bead surface is neutralized. Purified DNA is thus eluted into this elution buffer and can be adapted for PCR applications.

Invitrogen© iPrep™ ChargeSwitch® magnetic-bead specifications

Bead Binding Capacity: 5-10 μg genomic DNA per mg

Bead Size: < 1 μm

Bead Concentration: 25 mg/ml

Storage Buffer: 10 mM MES, pH 5.0, 10 mM NaCl, 0.1% Tween 20

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Thermo Scientific© Nanodrop 2000

The Thermo Scientific© Nanodrop 2000 is a micro-volume UV-VIS spectrophotometer its primary function application is nucleic acid and protein quantification for sample volumes as low as 0.5µl (Thermo Scientific©)

Stock Solution Dilution

A serial dilution is the spepwise approach adhered to for a dilution process relating to a substance in solution, with this instance it relating C.parvum oocysts in reagent water. The dilution factor remains constant, consequentially resulting in a geometric progression of the concentration in a logarithmic fashion. This procedure is utilized to generate an accurate, highly diluted solution for experimental procedures which require a concentration curve with a logarithmic scalce (Aneja, K. R. 2005).

Scientific Paper

Utilisation of a novel iPrep™ ChargeSwitch® Forensic kit as a means to increase the sensitivity of a Nested-PCR for the detection of DNA isolated from oocysts of Crytptosporidium parvum.

Cliff Gilligan.

Centre for Molecular Bioscience, University of Ulster, Cromore Road, Coleraine, County Londonderry BT52 1SA, Northern Ireland.

Abstract

In this investigation, a nested-PCR was utilized for the detection of Cryptosporidium parvum (C.parvum) at low quantities. This assay demonstrated a capability of reproducibly detecting oocysts at concentrations of less than or equal to 2.5oocysts per 50ul. Initially, under the experimental conditions a detection capability of 25,000 per 50ul was established. A dilution of the stock solution was implemented. Thus the lower oocyst concentration per sample would represent concentration levels similar to those which one would expect to find in contaminated water samples. Water-borne transmission of C.parvum has become far more widespread in the last decade. This experimental procedure integrated a novel Invitrogen© iPrep™ ChargeSwitch® Forensic kit for DNA isolation from C.parvum oocysts for use with a nested-PCR assay in the hope of increasing the sensitivity of the nested-PCR to detect low DNA concentration levels extracted from oocysts. The low levels of oocysts present in the samples examined made the selection of the nested-PCR assay the method of choice as it is reliable low level detection molecular technology adept at screening high numbers of potable water samples. In contrast to the conventional immune-fluorescence assay (IFA) method which is time-consuming, skilled labour intensive and liable to false positive and negative results the nested-PCR assay is more reproducible, cost effective and can easily distinguish between viable and non-viable C.parvum oocysts.

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Keywords: Cryptosporidium parvum; oocyst; iPrep™ ChargeSwitch® Forensic kit; stock dilution; nested-PCR

2.1 Introduction

The discovery of Cryptosporidium has been credited to Tyzzer in 1907, despite being one if not the most prevalent waterborne diseases in the world the first confirmed case of Cryptosporidiosis was not reported until 1976 (Morgan and Upton 2000; Santin and Zarlenga 2009). Cryptosporidium literally translates as "hidden spore" and is a coccidian parasite within the phylum Apicomplexa. Regarded as intracellular, obligate parasites which pertain and infect the lower part of the intestinal tract thereby causing diarrhorea in a plethora of vertebrate hosts including birds and animals (Morgan and Upton 2000; Monis and Saint 2000). Immuno-compromised patients specifically AID's afflicted individuals, suffer severe and often fatal symptoms from this disease.

To date twenty valid species and greater than 40 genotypes of Cryptosporidium have been identified thus far (Chalmers and Davis 2009). Xiao and Feng in 2005 cited five cryptosporidium species/genotypes as being responsible for the majority of human cryptosporidium cases which include C.parvum, C.meleagridiz, C.felis and C.canis (Xiao and Feng 2005; Fayer and Xiao 2008).

Within these species, macrogamonts and microgamonts display independent development. A macrogamont will give rise to abundance of male gametes and two morphofunctional types of oocysts allowing parasitic infection within various environmental conditions.

The innate ability of Cryptosporidium to infect a myriad of animals and because of the ubiquitous presence of cryptosporidium oocysts within the environment, numerous routes of transmission exist for humans to acquire cryptosporidium infection. These include direct contact with an infected individual (person to person transmission infection) or via animals (zoonotic transmission infection), ingestion of contaminated food (food borne infection transmission) or drinking infected water (water borne infection transmission) (Fayer and Xiao 2008).

C.parvum and C.hominis are responsible for greater than 90% of cryptosporidium cases in the western world with the remaining percentage attributed to C.meleagridis, C.canis or C.felis. Geographical location has a direct correlation on the distribution of C.parvum and C.hominis in humans with a possible explanation for such a phenomenon being, the routes of transmission into humans varies in different geographical locations thus the dominant species/genotype will also vary to (Xiao and Feng 2005; Fayer and Xiao 2008).

The cryptosporidium life cycle contains a spore phase (oocyst) enabling survival for long periods of time independent to a host. Resistance to disinfectants such as chlorine bleach based disinfectants ensures cryptosporidium oocysts remain highly prevalent with regards to infective capability over this independent period (http://www.cdc.gov). An asexual stage and a sexual stage are integrated into the life cycle of cryptosporidium parvum. Post ingestion, the oocysts excyst within the small intestinal tract. They release sporozoites that form an attachment to the microvilli of the epithelial cells within the small intestines. Sporozoites after attachment become trophozoites that reproduce asexually through multiple fission in a process known as schizogony. The trophozoites develop into type I meronts that contain 8 daughter cells and type II meronts which contain 4 type II merozoites (http://www.cdc.gov; Ryan et al., 2004). The merozites in question after being released form an attachment to the brush border of the epithelial cells where they diversify into (female) macrogamonts and (male) microgamonts (Chen et al., 2003). Microgamonts pertaining to macrogamonts form zygotes. Zygotes develop into two morphofunctional distinct forms of oocysts. Thick walled oocysts are excreted into the environment whereby they can survive within various environmental conditions for months (Chen et al., 2003).

Vital dye staining techniques have been developed to assess oocyst viability which pivot around staining with 4,'6-diamidino-2-phenylindole, dihydrochoride (DAPI) and propidium iodine (PI). However microscopy based methods cannot be deemed as amenable to high-throughput sample screening as a means of numerical determination as they are extremely high-skilled labour intensive (Laxer, M.A 1991).

The advent of the molecular era herald Polymerase Chain Reaction (PCR) as a viable alternative to the conventional methods available. The sensitivity detection level of this assay procedure varies from as little as 1 oocyst to several thousand oocysts and is highly dependent on the nucleic acid extraction technique employed on the sample matrix (Laxer, M.A 1991).

The Invitrogen© iPrep™ ChargeSwitch® Forensic kit technique permits quick ad effective purification of genomic DNA from forensic samples, in this instance the purified isolation of C.parvum DNA from C.parvum oocysts in solution. This novel approach utilizes magnetic bead based technology to purify and isolate the genomic DNA required for PCR applications without the use of hazardous chemicals, centrifugation or manifolds.

PCR-based methods have the potential to remedy many of the limitations associated with IFA. The advantages inherent to nested-PCR include a much greater specificity, greater sensitivity and a greater repeatability rate (Laxer, M.A 1991).

This investigation critiques the ability of a nested-PCR assay's ability for the detection of C.parvum DNA from low oocyst concentrations after the implementation of a novel Invitrogen© iPrep™ ChargeSwitch® Forensic kit technique. This technique represents a new method of DNA isolation from oocysts. The effectiveness of this procedure on low oocyst concentrations was assessed by implementing a series dilution of a stock solution containing C.parvum DNA extracted by this novel method with the lowest dilution rate representing 2.5 oocysts per 50µl of solution. This study suggests that the series dilution would represent oocyst concentration levels one would expect to find in contaminated water samples.

Materials and Methods

Source and preparation of Cryptosporidium parvum oocysts

The stock solution of C.parvum utilized in this investigation had previously been acquired from (Invitrogen CA, United States). The viability of the concentration had previously been determined by utilizing vital dye staining 4,'6-diamidino-2-phenylindole, dihydrochoride (DAPI) and propidium iodine (PI) and numerical determination of the oocyst concentration had been established by a Nanodrop 2000 (Thermo Fischer Scientific, United States) as outlined in the protocol by (Balkis, 2010) as part of her PhD study. Working from information inherent to this PhD study, it was first necessary to establish that the nested-PCR technique, which formed a major aspect of this investigation, was adept at detecting C.parvum DNA isolated from C.parvum oocysts via a P.A.L.M Laser Capture Micro-dissection (LCM) cutting and catapulting technique prior to the introduction of a novel Invitrogen© iPrep™ ChargeSwitch® Forensic kit technique. This new technique represented a new method for C.parvum DNA isolation from C.parvum oocysts which were present in the C.parvum stock solution (Invitrogen CA, United States) at a ratio of 10, 000,000 per 1ml.

Demonstration of Nested-PCR assays ability to detect a C.parvum DNA sample extracted from oocysts by the P.A.L.M Laser Capture Micro-dissection (LCM)

Initially in order to establish that the Nested-PCR was working within normal operational parameters a nested-PCR was carried out on a C.parvum DNA sample which had been previously extracted from C.parvum oocysts by the established P.A.L.M LCM technique. This DNA sample was obtained from a stock solution of C.parvum (Invitrogen© CA, United States). This sample had underwent purification, numerical determination and viability testing as outlined in the protocol by (Balkis, 2010 ) as part of her PhD study and therefore could act as an internal positive control (IPC) for future nested-PCR's within this investigation, which utilized the novel Invitrogen© iPrep™ ChargeSwitch® Forensic kit DNA extraction technique.

The nested-PCR for this DNA sample was orchestrated in sequential reactions. Both the primary and secondary PCR aspects of this nested-PCR were carried out on a Techne TC-500 PCR system (Techne©, Cambridge, UK) under the following procedural conditions: 94°C for 3minutes; followed by 35 cycles of the following 94°C for 45seconds, 55°C for 45seconds, 72°C for 1minute and then finally 72°C for 7minutes which was the final extension period. Then an unlimited hold period was established at 4°C.

The primers utilized in the primary PCR were Invitrogen© primers

F1 (5'-TTCTAGAGCTAATACATGCG-3') and

R1 (5'-CCCATTTCCTTCAAACAGCA-3')

The secondary PCR primers were also Invitrogen© primers

F2 (5'-GGAAGGGTTGTATTTATTAGATAAAG-3') and

R2 (5'-AAGGAGTAAGGAACAACCTCCA-3')

In the primary PCR, two reaction master-mixes were used respectively, the first containing a total concentration of (1ml) and the other containing a total concentration of (500µl). The (1ml) master-mix contained the following; 100µl of 10x PCR buffer, 90µl of 50Mm MgCl2, a total of 8µl of deoxynucleoside triphosphates, [2µl each of d(ATP), d(TTP), d(GTP) d(CTP) ], 25µl of primer (F1) and 25µl of primer (F2), 732µl of double distilled (dd) H2O, 10µl of Taq and 10µl of sample (C.parvum DNA).

The 500ml master-mix contained the following; 50µl of 10x PCR buffer, 45µl of 50Mm MgCl2, a total of 4µl of deoxynucleoside triphosphates, [ 1µl each of d(ATP), d(TTP), d(GTP) d(CTP) ], 25µl of primer (F1) and 25µl of primer (F2), 331µl of double distilled (dd) H2O, 10µl of Taq and 10µl of sample (C.parvum DNA).

The secondary PCR aspect of this procedure also had two reaction master-mixes, the first again containing a total concentration of (1ml) and the other containing (500µl). The (1ml) master-mix contained the following; 100µl of 10x PCR buffer, 30µl of 50Mm MgCl2, a total of 8µl of deoxynucleoside triphosphates, [ 2µl each of d(ATP), d(TTP), d(GTP) d(CTP) ], 50µl of primer (F1) and 50µl of primer (F2), 732µl of double distilled (dd) H2O, 10µl of Taq and 20µl of sample (Primary PCR product).

The 500ml master-mix contained the following; 50µl of 10x PCR buffer, 15µl of 50Mm MgCl2, a total of 4µl of deoxynucleoside triphosphates, [ 1µl each of d(ATP), d(TTP), d(GTP) d(CTP) ], 50µl of primer (F1) and 50µl of primer (F2), 30µl of double distilled (dd) H2O, 10µl of Taq and 20µl of sample (Primary PCR product).

Amplification products were detected by electrophoresis of agarose gels containing ethidium bromide (Sigma United Kingdom) adhering to standard laboratory operational procedure (SOP's).

Implementation of novel Invitrogen© iPrep™ ChargeSwitch® Forensic kit for nucleic acid extraction

100µl of C.parvum oocyst stock solution was pipetted into a sterile 1.5ml locking cap micro-centrifuge tube adhering to aseptic technique within a fume-hood.

Preparation of the sample for purification

Adhering to the standard operational procedure (SOP) of the Invitrogen© iPrep™ ChargeSwitch® Forensic kit manual, 15 freeze thaws of the sample were performed by carefully placing the 1.5-ml locking-cap micro-centrifuge tube into liquid nitrogen (LN) for 1minute, them removing with thongs and placing in a heat block set to 65°C for 1minute. After the freeze-thaws were carried out, the tube was allowed to stand at room temperature for 2minutes. An addition of 10µl of Proteinase K (Invitrogen CA, United States) was made by pipette and then the sample was placed in a heat block for 1.5hours at 55°C.

After the incubation period was complete, the tube was carefully opened and Lysis Buffer (Invitrogen CA, United States) was added to bring the final concentration volume to 1ml.

Binding the DNA

Next 200µl of Purification Buffer (Invitrogen CA, United States) was added to the sample followed by an addition of 20µl of Invitrogen© iPrep™ ChargeSwitch® Magnetic beads. A gentle pipette tip mix was adhered to 5times to ensure an even suspension of the magnetic beads was achieved. The sample was incubated at room temperature for 5minutes to allow the DNA to efficiently bind to the beads with a gentle pipette mix occurring 2.5 minutes into the incubation period. The tube was then placed on the MPC-S magnet, with the magnet in the vertical position, until the beads formed a tight pellet and the supernatant had cleared. This took on average 55seconds. Subsequently it was then necessary to aspirate and discard the supernatant without removing the tube from the magnet or disturbing the pellet of beads. This was achieved by angling the pipette in such a manner that the tip was pointed away from the pellet.

Washing the DNA

The tube was removed from the magnet and it was noted that there was no supernatant left in the tube which was visible to the naked eye. An addition of 500µl of Wash Buffer (Invitrogen CA, United States) was made to the tube. Using a 1-ml pipette to 300µl, a gentle pipette tip mix was carried out 5times to re-suspend the magnetic beads efficiently again in a manner which ensured an even disruption. The tube was then placed back onto the magnet for precisely 1minute which allowed the beads to form a tight pellet and the supernatant was clear. Again without removing the tube from the magnet, the supernatant was aspirated and discarded without disturbing the pellet by angling the pipette in such a manner that the pipette tip did not disturb the pellet. The tube was then separated from the magnet.

Eluting the DNA

After the tube was detached from the magnet, an inspection of the tube with the naked eye detected no supernatant present in the tube An addition of 50µl of elution buffer was made to the tube and the solution was pipette mixed up and down gently 10 times to efficiently re-suspend the magnetic beads. The elution buffer was pre-warmed to 60°C before addition to increase the DNA yield. The sample was then reattached to the magnet for 1minute to allow the beads to form a rigid pellet and the supernatant turned clear. Without separating the sample from the magnet, the now clear supernatant which contained the C.parvum DNA was transferred to a sterile micro-centrifuge tube without unsettling the pellet. This was achieved by angling the pipette in a manner that was away from the pellet. The magnetic beads were degraded as they were not reusable. The purified DNA was stored at -20°C until required for the nested-PCR procedure.

Nested PCR on non-diluted nucleic acid extraction Invitrogen© iPrep™ ChargeSwitch® Forensic kit nucleic acid extraction samples.

The next procedural step entailed performing a nested-PCR on the C.parvum DNA which had underwent extraction via the novel Invitrogen© iPrep™ ChargeSwitch® Forensic kit nucleic acid procedure. This nested-PCR would confirm or reject the validity of the novel Invitrogen© iPrep™ ChargeSwitch® Forensic kit approach as an alternative means of DNA extraction to the established P.A.L.M LCM technique. This nested-PCR was again orchestrated in sequential reactions. Both the primary and secondary PCR aspects of this nested-PCR were carried out on a Techne TC-500 PCR system (Techne©, Cambridge, UK) under the same procedural conditions outlined in section 2.3 in materials and methods. The Invitrogen© primers were also the same as discussed in section 2.3 of materials and methods.

The primary PCR aspect of this nested-PCR was altered to only utilize the (1ml) reaction master-mix. The 1ml master-mix contained the same volume concentrations as discussed in section 2.3 of the materials and methods.

This nested-PCR contained DNA samples which had underwent different DNA extraction techniques. The DNA samples extracted by the P.A.L.M LCM technique were utilized as IPC's and would allow a direct comparison to be drawn between the amplification products of the two different nucleic acid extraction techniques used in this nested-PCR.

Series dilution of stock solution

A series dilution of the stock solution of C.parvum DNA which was extracted by the Invitrogen© iPrep™ ChargeSwitch® Forensic kit was performed. This was carried out to replicate samples one would expect to examine from contaminated potable water. The purchased stock solution of C.parvum oocysts contained 10,000,000 per 1ml of solution thus an extraction of 2.5µl of stock solution would contain 25,000 oocysts. 2.5µl of stock solution was transferred into 47.5µl of reagent water creating a solution which would act as a new primary stock solution for this series dilution with a ratio of 25,000 oocysts per 50µl of solution. 1µl of this solution which contained 25,000 C.parvum oocysts was then transferred to 49µl of reagent water creating a solution containing a solution containing 2,500 oocysts per 50µl of solution. This procedure was replicated three more times until a solution was created which contained 2.5 C.parvum oocysts per 50µl of solution.

Nested PCR on stock dilution series of nucleic acid extraction from Invitrogen© iPrep™ ChargeSwitch® Forensic kit nucleic acid extraction samples.

The final procedural step entailed performing a nested-PCR on the C.parvum DNA extracted by the Invitrogen© iPrep™ ChargeSwitch® Forensic kit which had underwent a series dilution. This PCR would accurately assess the detection capability of the nested-PCR procedure on vastly reduced numbers of C.parvum oocysts in essence testing the sensitivity limits of the assay. The C.parvum DNA samples used were extracted from a primary stock solution containing 25,000 per 50µl (which had underwent the novel Invitrogen© DNA extraction approach) and then diluted until a suspension was created which represented 2.5 C.parvum oocysts per 50 µl of solution.

This nested-PCR again adhered to a sequential PCR format on the Techne©, TC-500 PCR system (Techne©, Cambridge UK) and used the same primers and procedural steps as described in section 2.3 of the materials and methods. (This nested-PCR procedure was carried out in duplicate.

Results and Discussion

The results inherent to this investigation pertain essentially to various nested-PCR's which utilized C.parvum DNA extracted from C.parum oocysts via two separate extraction methods; DNA extraction by the P.A.L.M LCM cutting and catapulting technique and the Invitrogen© iPrep™ ChargeSwitch® Forensic kit technique.

Firstly a nested-PCR was performed on C.parvum DNA acquired by the already proven P.A.L.M LCM DNA extraction technique. Secondly having introduced a novel method of DNA extraction, the Invitrogen© iPrep™ ChargeSwitch® Forensic kit technique, a nested-PCR was executed on the DNA sample acquired by this technique. This nested-PCR also contained DNA samples acquired from the P.A.L.M LCM technique and acted as IPC's and allowed a comparison to be drawn between the various technique amplification products. Finally a dilution series was performed on the DNA sample attained from Invitrogen© iPrep™ ChargeSwitch® Forensic kit after which a final set of nested-PCR's were orchestrated on the samples in the dilution series which allowed sensitivity limits for this PCR assay to be scrutinized.

Detection of C.parvum by nested-PCR on a DNA sample extracted using the P.A.L.M Laser Capture Micro-dissection (LCM) technique.

A nested-PCR assay protocol for C.parvum DNA was adhered to, it utilized Invitrogen© F1 and R1 primers in the primary round of PCR and Invitrogen© F2 and R2 primers for the secondary round of the nested-PCR procedure. The Invitrogen© primers were designed to amplify a 590bp fragment from the C.parvum DNA (human genotypes tested) in the primary round of PCR and a 330bp fragment in the secondary round of PCR.

This assay detected C.parvum DNA extracted from C.parvum oocysts which were extracted using P.A.L.M LCM DNA technique. As detailed in section 2.3 (Materials and Methods) two distinct PCR reaction master-mixes were employed for amplification purposes.

From an examination of Fig 1.0 a determination can clearly be made as to which PCR reaction master-mix allowed for a positive detection of C.parvum DNA. The (1ml) total volume concentration PCR reaction master-mix clearly displayed a positive C.parvum detection result. Two strong amplication products were visible in the agarose gel lanes 3 and 5. In contrast lanes 2 and 4 (which contained the 500µl PCR reaction master-mix) displayed no amplification products.

Detection of C.parvum DNA by nested-PCR on a non-diluted Invitrogen© iPrep™ ChargeSwitch® Forensic kit nucleic acid extraction sample

Following certification that the P.A.L.M LCM technique was working after a successful examination of the nested-PCR assays detection capability on C.parvum DNA extracted from oocysts, it was then necessary to trial this technique on C.parvum DNA which has been extracted by the novel Invitrogen© iPrep™ ChargeSwitch® Forensic kit.

The same nested-PCR assay protocol which had proven successful for the detection of C.parvum DNA extracted by the P.A.L.M LCM technique was adhered to again. It used the Invitrogen© F1 and R1 primers in the primary round of PCR and Invitrogen© F2 and R2 primers for the secondary round of PCR. Again the Invitrogen© primers would display amplification products for a 590bp fragment from the C.parvum DNA (human genotypes tested) and a 330bp fragment in the primary PCR stage of the nested-PCR procedure.

The successful 1ml PCR reaction master-mix was only utilized this time on two separate C.parvum DNA samples. Lanes 2 and 3 contained C.parvum DNA extracted by the P.A.L.M LCM technique. Lanes 4, 5 and 6 containg C.parvum DNA samples extracted using the Invitrogen© iPrep™ ChargeSwitch® Forensic kit. The C.parvum DNA extracted by the P.A.L.M LCM technique would act as IPC's and allow a comparison to be drawn between the amplified products of the two different nucleic acid extraction techniques. In order to determine that the Invitrogen© primers did indeed amplify products for a 590bp fragment from the C.parvum DNA after the primary PCR, lanes 8, 9 and 10 contained C.parvum DNA extracted using the Invitrogen© iPrep™ ChargeSwitch® Forensic kit amplified only by the primary round of nested-PCR.

An examination of Fig 2.0 would suggest that both nucleic acid extraction techniques proved applicable to amplification via the nested-PCR. Strong amplification products were visible in lanes 2 and 3, which acted as positive controls. Lanes 4, 5 and 6 also displayed strong bands at the 330bp amplification mark. While all amplification products from the two different nucleic acid extraction techniques provided strong PCR bands, the bands in question [lanes 2, 3, 4, 5 and 6] were all deemed "messy" suggesting over amplification of the DNA sample which marked a promising result with regards to detecting amplification products after a series dilution was made of the C.parvum sample extracted by the Invitrogen© iPrep™ ChargeSwitch® Forensic kit.

In order to determine that the primary PCR was indeed operational for a 590bp fragment of the C.parvum DNA, Lanes 9, 10 and 11 contained Invitrogen© iPrep™ ChargeSwitch® Forensic kit extracted DNA only amplified by the primary round of the nested-PCR.

A general examination of lanes 9, 10 and 11 in Fig 2.0 displays clear amplification bands in lanes 10 and 11 and a faint amplification product visible in lane 9 at the 590bp amplification mark suggesting the primary round of PCR in the nested-PCR procedure was operating within normal parameters.

Detection of C.parvum by nested-PCR on dilution series of Invitrogen© iPrep™ ChargeSwitch® Forensic kit nucleic acid extraction samples.

This nested-PCR was carried out in duplicate and the results pertain to Fig 3.0 and Fig 4.0. The duplication procedure adapted allowed for a determination of applicability and reproducibility of this assay to be drawn. In both Fig 3.0 and Fig 4.0, lane 2 contained a C.parvum sample extracted from 25,000 oocysts per 50µl, lane 3 contained a C.parvum DNA sample extracted from 2,500 oocysts per 50µl, lane 4 contained a C.parvum DNA sample extracted from 250 oocysts per 50µl, lane 5 contained a C.parvum DNA sample extracted from 25 oocysts per 50µl and finally lane 6 contained a C.parvum DNA sample extracted from 2.5 oocysts per 50µl.

An examination of Fig 3.0 clearly indicates a detection of C.parvum DNA in lanes 2, 5 and 6. In contrast a negative detection result was viewed for lanes 3 and 4. As there was a clear detection for C.parvum DNA extracted from 2.5 oocysts per 50µl(the lowest inoclum level) this would suggest an operational error or sampling error was responsible for the negative results in lanes 3 and 4.

Fig 4.0 provided evidence that a nested-PCR utilizing C.parvum DNA extracted by the Invitrogen© iPrep™ ChargeSwitch® Forensic kit was highly sensitive, as a positive detection result was again viewed in lane 6. Lane 6 as previously stated represented the lowest inoclum level of C.parvum DNA. The presence of two strong amplification products in these lanes indicates that this procedure is reproducible to a low level of sensitivity.

The nested-PCR assay examined in Fig 4.0 in fact displayed distinctive strong bands in all lanes [2 to 7] thus implying that the Invitrogen© iPrep™ ChargeSwitch® Forensic kit was highly applicable to this nested-PCR procedure and as a result it can be deemed as highly efficient at removing PCR inhibitors existing in the various DNA samples.

Conclusion

As we enter an era where water resources will diminish and clean water supplies will be placed under extra pressure from a surge in demand it is now of the upmost importance to detect water-borne pathogens within this water supply chain. This need has directly resulted in a myriad of PCR based molecular assays coming on-line within recent years for the detection of cryptosporidium in water sources ( as reviewed by Wiedenmann et al., 1998).

Amplification of a target gene by PCR allows accurate detection of low numbers of cryptosporidium oocysts .This investigation was centered around a nested-PCR technique which integrated C.parvum DNA samples acquired by two separate nucleic acid extraction techniques; the P.A.L.M LCM cutting and catapulting technique and the Invitrogen© iPrep™ ChargeSwitch® Forensic kit.

The Invitrogen© iPrep™ ChargeSwitch® Forensic kit represented an alternative DNA extraction method to the established P.A.L.M LCM technique for nested-PCR detection of C.parvum oocysts in water samples. The results inherent to this investigation demonstrate that the nested-PCR can reliably detect C.parvum DNA extracted via the Invitrogen© iPrep™ ChargeSwitch® Forensic kit from as low an inoclum level as 2.5 oocysts spiked into reagent water.

Very few studies centered on the accurate detection of C.parvum have produced such accurate data on assay sensitivity and reproducibility. In an investigation by Kruger et al., 1998 his team confirmed a success detection rate of 25% for the accurate detection of 10 oocysts and the 100% detection rate of 20 oocysts spiked into reagent water. This investigation reports a 100% success detection rate for the detection of 2.5 oocysts spiked into reagent water. Albeit this experiment procedure was only carried out in duplicate, the results inherent proved extremely positive for the use of the Invitrogen© iPrep™ ChargeSwitch® Forensic kit as a means of purifying DNA to increase the nested-PCR's sensitivity and reproducibility.

When this study ran nested-PCR's on C.parvum DNA extracted by the two nucleic acid extraction techniques under investigation,the nested-PCR amplification product results displayed very little difference thus suggesting both techniques of DNA extraction were highly applicable to use with nested-PCR. An examination of the primary-PCR amplification products displayed "clean" strong bands suggesting amplification products even at this early stage indicated the Invitrogen© iPrep™ ChargeSwitch® Forensic kit extraction technique was working at a very successful level and has highly adept at removing PCR inhibitors existing within the DNA samples.

This conclusion with regards the purification capability of the Invitrogen© iPrep™ ChargeSwitch® Forensic kit for the removal of PCR inhibitors is also backed up by the fact that when this Invitrogen© kit was used on the series dilution of C.parvum DNA the nested-PCR assay could readily detect C.parvum oocysts spiked into reagent water achieving a detection limit for as few as 2.5 oocysts per 50µl of solution. The sensitivity capability of this Invitrogen© kit will truly be realised in future research when environmental water samples are utilized, such samples will contain higher organic and inorganic components as well as environmental protein and debris. This Invitrogen© kit will come to the fore as the desired DNA extraction technique AS its purification ability will make it highly feasible to detect low concentrations of C.parvum DNA in highly dilute environmental water samples.

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