Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-06-04
2002-08-20
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S325000, C435S091100, C435S091200, C435S007210, C435S007220, C435S091400, C435S091500, C435S091510, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06436638
ABSTRACT:
BACKGROUND
Protozoan parasites are a major cause of gastrointestinal disease. Within the last decade, the protozoa Cryptosporidium and Giardia have been increasingly associated with waterborne outbreaks of acute diarrhea.
Cryptosporidium parvum
is of particular concern because no known treatment of the illness is available at present. Moreover, in the immunocompromised host, a
C. parvum
infection can lead to prolonged severe diarrhea, malnutrition, wasting, and death.
Cryptosporidium is an enteric coccidia, which has a multi-staged life cycle one to eight days in duration. The oocyst contains four sporozoites which, during normal infection, are released in the presence of bile salts and proteases. The sporozoites attach and penetrate intestinal epithelial cells. Once inside, they develop into a rounded trophozoite in the area between the cytoplasmic membrane and the cytoplasm. Through asexual reproduction, the trophozoite (a type I meront) forms up to eight merozoites. The merozoites may then develop into a type II meront, which, by asexual reproduction, forms four merozoites. The second generation merozoites may develop into male (microgamont) or female (macrogamont) forms. The male form may lead to the sexual phase of the Cryptosporidium life cycle which culminates, in vivo, in the production of the environmentally resistant oocysts. These hardy structures possess a thick, double-layered protective cell wall which is resistant to most disinfectants, chlorine concentrations generally present in municipal water supplies, and temperatures between −4° C. and 42° C.
Cryptosporidium is prevalent in most vertebrate groups. Domestic animals, such as rodents, kittens, puppies, and calves may constitute an important reservoir of the human Cryptosporidium. However, disease outbreaks in day-care centers, hospitals and urban family groups indicate that most human infections are transmitted person-to-person rather than via a zoonotic route. Since oocysts are found almost exclusively in stool, the transmission is undoubtedly fecal-oral. Moreover, the recovery of oocysts from both surface and drinking water suggests that indirect transmission via water is not uncommon.
Quantitative studies on the infectious dose for humans are currently limited. One study found that, in healthy volunteers, the infectious dose (ID
50
) is 132 oocysts, with as few as 30 oocysts causing infection in 20% of individuals tested (DuPont et al., 1995). However, the ID
50
could be lower, such as one to ten oocysts, in more susceptible individuals.
Indeed, Cryptosporidium has been documented as a major cause of waterborne illness on numerous occasions. The largest outbreak occurred during the spring of 1993 in Milwaukee, Wis., resulting in approximately 400,000 illnesses and 100 deaths (MacKenzie et al., 1994).
Over the last 10 years, Cryptosporidium oocysts have been found in 9.1 to 100% of surface waters tested at concentrations ranging from 0.003 to 1,920 oocysts per liter. Oocysts were also detected in 27% and 17% of finished water samples in two multi-state surveys.
These studies, surveys, and documented outbreaks clearly indicate that infectious Cryptosporidium may be found in source water and the efficiency of conventional water treatment needs to be closely monitored. Indeed, the occurrence of the causative agents
Cryptosporidium parvum
and
Giardia lamblia
in water supplies has become a critical issue for the water industry.
The current techniques for isolating Cryptosporidium and Giardia from water involve filtration and centrifugation to concentrate and purify oocysts and cysts, respectively, followed by immunofluorescence microscopy. Objects with the correct shape, dimensions, and fluorescence are confirmed by observation of internal structures using differential interference contrast microscopy. The limitations of these procedures include loss of oocysts or cysts during isolation, resulting in recovery efficiencies ranging from 100 percent to less than one percent for Cryptosporidium. Moreover, the immunofluorescent assay (IFA) method cannot distinguish viable and potentially infective from non-viable or non-infective oocysts and cysts. Additional limitations of IFA include nonspecific antibody binding and cross-reactive antibody binding among human and animal infective species of Cryptosporidium or Giardia.
For the foregoing reasons, there is a need for an alternative method of detecting Cryptosporidium and Giardia pathogens that is rapid, sensitive, and specific. Moreover, the alternative method would be able to determine if Cryptosporidium oocysts are viable and infective.
SUMMARY
The present invention includes a method for selectively detecting the presence of
C. parvum
organisms in a sample potentially containing
C. parvum
organisms and other Cryptosporidium organisms. The method comprises, first, selectively amplifying at least a portion of
C. parvum
HSP70 polynucleotide present in the sample using a primer, and then, detecting the presence of any amplified polynucleotide formed. The presence of amplified polynucleotide indicates the presence of
C. parvum
organisms in the sample. The method can additionally comprise recovering
C. parvum
oocysts from the sample, prior to amplifying the
C. parvum
polynucleotide. Further, the method can additionally comprise extracting
C. parvum
DNA from the recovered oocysts, prior to amplifying the
C. parvum
polynucleotide.
In a preferred embodiment, the primer has a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. In another preferred embodiment, the amplification is performed by a temperature cycling amplification reaction, such as a polymerase chain reaction. Alternately, the amplification is performed by an isothermal amplification reaction, such as a self-sustained sequence replication reaction.
In a preferred embodiment, the detecting is performed by subjecting the amplified polynucleotide to hybridization conditions with a DNA probe or with a PNA probe, such as a probe having a sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO: 10, the complement of SEQ ID NO:9 and the complement of SEQ ID NO:10.
The present invention also includes a method for selectively detecting the presence of
C. parvum
organisms and for detecting the presence of
G. lamblia
organisms, simultaneously, in a sample potentially containing
C. parvum
organisms and
G. lamblia
organisms, and where the sample also potentially contains other Cryptosporidium species organisms. The method comprises, first, amplifying at least a portion of the
G. lamblia
HSP polynucleotide present in the sample using a first primer, and selectively amplifying at least a portion of the
C. parvum
HSP70 polynucleotide-present in the sample using a second primer, and then detecting the presence of any amplified polynucleotide formed. The presence of amplified
C. parvum
HSP70 polynucleotide indicates the presence of
C. parvum
organisms in the sample, and the presence of amplified
G. lamblia
HSP polynucleotide indicates the presence of
G. lamblia
organisms in the sample. The method can additionally comprise recovering
C. parvum
oocysts or
G. lamblia
cysts from the sample, prior to amplifying the
C. parvum
polynucleotide. Further, the method can additionally comprise extracting
C. parvum
DNA from the recovered oocysts or
G. lamblia
DNA from the recovered cysts prior to amplifying the
C. parvum
polynucleotide or the
G. lamblia
polynucleotide.
In a preferred embodiment, the first primer has a sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:8, and the second primer has a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. In another preferred embodiment, the amplification is performed by a temperature cycling amplification reaction, such as a polymerase chain reaction. Alternately, the amplification is performed by an isothermal amplification reaction, such as a self-sustained sequence replication reaction.
In a preferred embodiment, the detec
De Leon Ricardo
Rochelle Paul A.
Collett James W.
Farah David A.
Guzo David
Metropolitan Water District of Southern California
Sheldon & Mak
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