scholarly journals Species-Specific, Nested PCR-Restriction Fragment Length Polymorphism Detection of Single Cryptosporidium parvum Oocysts

2001 ◽  
Vol 67 (6) ◽  
pp. 2665-2668 ◽  
Author(s):  
Gregory D. Sturbaum ◽  
Carrie Reed ◽  
Paul J. Hoover ◽  
B. Helen Jost ◽  
Marilyn M. Marshall ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Cryptosporidium Parvum ◽  
Restriction Enzyme ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Nested Pcr ◽  
Fragment Length Polymorphism ◽  
Enzyme Digestion ◽  
Restriction Enzyme Digestion ◽  
Restriction Fragment Length

ABSTRACT Concurrent with recent advances seen with Cryptosporidium parvum detection in both treated and untreated water is the need to properly evaluate these advances. A micromanipulation method by which known numbers of C. parvum oocysts, even a single oocyst, can be delivered to a test matrix for detection sensitivity is presented. Using newly developed nested PCR-restriction fragment length polymorphism primers, PCR sensitivity was evaluated with 1, 2, 3, 4, 5, 7, or 10 oocysts. PCR detection rates (50 samples for each number of oocysts) ranged from 38% for single oocysts to 92% for 5 oocysts, while 10 oocysts were needed to achieve 100% detection. The nested PCR conditions amplified products from C. parvum,Cryptosporidium baileyi, and Cryptosporidium serpentis but no other Cryptosporidium sp. or protozoan tested. Restriction enzyme digestion with VspI distinguished between C. parvum genotypes 1 and 2. Restriction enzyme digestion with DraII distinguishedC. parvum from C. baileyi and C. serpentis. Use of known numbers of whole oocysts encompasses the difficulty of liberating DNA from the oocyst and eliminates the standard deviation inherent within a dilution series. To our knowledge this is the first report in which singly isolatedC. parvum oocysts were used to evaluate PCR sensitivity. This achievement illustrates that PCR amplification of a single oocyst is feasible, yet sensitivity remains an issue, thereby illustrating the difficulty of dealing with low oocyst numbers when working with environmental water samples.

scholarly journals Cryptosporidium parvum Mixed Genotypes Detected by PCR-Restriction Fragment Length Polymorphism Analysis

2002 ◽  
Vol 68 (1) ◽  
pp. 427-429 ◽  
Author(s):  
Carrie Reed ◽  
Gregory D. Sturbaum ◽  
Paul J. Hoover ◽  
Charles R. Sterling
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Cryptosporidium Parvum ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Polymorphism Analysis ◽  
Genotype I ◽  
Single Genotype ◽  
Restriction Fragment Length

ABSTRACT Combinations of 10 Cryptosporidium parvum oocysts, with various ratios of genotype I to genotype II, were isolated and subjected to PCR-restriction fragment length polymorphism analysis. Amplification of both genotypes in these samples ranged from 31 to 74% and yielded no information about the genotype proportions. In addition, since both genotypes were not always detected, amplification of a single genotype is not conclusive evidence that the sample contains only a single genotype.

scholarly journals Restriction fragment length polymorphism analysis of genes of virulent strain isolate of Toxoplasma gondii using enzyme DdeI

2021 ◽  
pp. 196-203
Author(s):  
Fitrine Ekawasti ◽  
Umi Cahyaningsih ◽  
N. L. P. Indi Dharmayanti ◽  
Siti Sa'diah ◽  
Didik Tulus Subekti ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Restriction Fragment Length Polymorphism ◽  
Genetic Markers ◽  
Restriction Enzyme ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Virulent Strain ◽  
Pcr Rflp ◽  
Restriction Fragment Length

Background and Aim: Toxoplasma gondii is a unicellular coccidian parasite distributed globally and is an important zoonotic pathogen. Approximately 30% of the human population worldwide is chronically infected with T. gondii. The pathogenicity of this species depends on the type originating from the clonal population. Techniques for more accurately determining the type of T. gondii have recently been developed using genetic markers. Specifically, T. gondii has been typed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). This study aimed to identify sets of PCR-RFLP markers that have high power to discriminate genotyping of T. gondii and are easy to use and are easy to use. The objective of this study was to characterize virulent strain isolates of T. gondii by PCR-RFLP using 10 markers with DdeI. Materials and Methods: T. gondii tachyzoites (RH virulent strain) were derived from culture cells at the Indonesian Research Center for Veterinary Sciences. Genotyping was performed on T. gondii DNA extracted from cell cultured tachyzoites using 10 genetic markers of PCR-RFLP, namely, B1#1, B1#2, B1#3, SAG1#1, SAG1#2, P30, BAG1, ROP1, GRA1, and GRA7, with digestion using the restriction enzyme DdeI. Results: The 10 genes were amplified by PCR. Among them, three genetic markers, B1#3, ROP1, and GRA1, were genotyped by the PCR-RFLP using restriction enzyme DdeI. Overall, the findings showed that the specific RFLP profile of digestion of gene regions by DdeI could be used as a specific marker for the virulent biotype causative of toxoplasmosis. In addition, virulent strains of T. gondii can be easily detected by these markers. Conclusion: Three pairs of primers (B1#3, ROP1, and GRA1) with DdeI have proven useful for the diagnosis of acute toxoplasmosis (virulent strain biotype I). This proposed method is relatively simple, rapid, cheap, and can be performed in most laboratories, providing a practical approach for the routine analysis of T. gondii strains.

Predominance of Cryptosporidium parvum genotype among diarrheic children from Egypt as an indicator for zoonotic transmission

2014 ◽  
Vol 60 (1) ◽  
Author(s):  
Maysa Ahmad Eraky ◽  
Azza Mohammed-Salah El-Hamshary ◽  
Hassan Hassan Hamadto ◽  
Kareem Fetouh Abdallah ◽  
Wafaa Moustafa Abdel-Hafed ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Restriction Fragment Length Polymorphism ◽  
Cryptosporidium Parvum ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Chain Reaction ◽  
Cryptosporidium Spp ◽  
Polymerase Chain ◽  
Restriction Fragment Length

AbstractCryptosporidium is a genus of zoonotic pathogens transmissible from a variety of animals to humans and is a considerable public health concern. It is a significant cause of diarrheal disease in developing and industrialized nations. Cryptosporidium parvum and Cryptosporidium hominis are the main agents of cryptosporidiosis in humans. In this study we identified the genotypes of the Cryptosporidium isolates from clinical samples from diarrheic children using polymerase chain reaction (PCR) amplification and restriction fragment length polymorphism (RFLP) analyses of the TRAP-C2 gene (Thrompodin Related Adhesive Protein). A total of 430 fecal specimens from 1 to 14 years children were collected from inpatient and outpatient clinics of Benha University, Educational and Children Specialized Hospitals, Benha, Qalubyia, and were microscopically examined for Cryptosporidium spp. All infected samples were also analyzed using nested PCR. A polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the (266-366 bp) of TRAP-C2 gene was also used to detect and identify Cryptosporidium spp. in PCR- positive samples. The results showed that 50 (11.63%) of the specimens were positive for Cryptosporidium spp. Genomic amplification and restriction digestion of the PCR products by BstETI, Hae III for TRAP-C2 gene restriction enzymes revealed that 82% (41/50) had C. parvum, 12% (6/50) had C. hominis, and three (3/50) samples (6%) had mixed infections. In conclusion, elevated prevalence of C. parvum, suggesting animal-human (zoonotic) transmission and further investigations are required to determine the subgenotypes of C. parvum to clarify the mode of transmission in order to improve the control measures.

scholarly journals Detection and Resolution of Cryptosporidium Species and Species Mixtures by Genus-Specific Nested PCR-Restriction Fragment Length Polymorphism Analysis, Direct Sequencing, and Cloning

2011 ◽  
Vol 77 (12) ◽  
pp. 3998-4007 ◽  
Author(s):  
Norma J. Ruecker ◽  
Rebecca M. Hoffman ◽  
Rachel M. Chalmers ◽  
Norman F. Neumann
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Nested Pcr ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Content Type ◽  
Pcr Rflp ◽  
Pcr Products ◽  
Restriction Fragment Length

ABSTRACTMolecular methods incorporating nested PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene ofCryptosporidiumspecies were validated to assess performance based on limit of detection (LoD) and for detecting and resolving mixtures of species and genotypes within a single sample. The 95% LoD was determined for seven species (Cryptosporidium hominis,C. parvum,C. felis,C. meleagridis,C. ubiquitum,C. muris, andC. andersoni) and ranged from 7 to 11 plasmid template copies with overlapping 95% confidence limits. The LoD values for genomic DNA from oocysts on microscope slides were 7 and 10 template copies forC. andersoniandC. parvum, respectively. The repetitive nested PCR-RFLP slide protocol had an LoD of 4 oocysts per slide. When templates of two species were mixed in equal ratios in the nested PCR-RFLP reaction mixture, there was no amplification bias toward one species over another. At high ratios of template mixtures (>1:10), there was a reduction or loss of detection of the less abundant species by RFLP analysis, most likely due to heteroduplex formation in the later cycles of the PCR. Replicate nested PCR was successful at resolving many mixtures ofCryptosporidiumat template concentrations near or below the LoD. The cloning of nested PCR products resulted in 17% of the cloned sequences being recombinants of the two original templates. Limiting-dilution nested PCR followed by the sequencing of PCR products resulted in no sequence anomalies, suggesting that this method is an effective and accurate way to study the species diversity ofCryptosporidium, particularly for environmental water samples, in which mixtures of parasites are common.

scholarly journals PCR-Restriction Fragment Length Polymorphism Analysis of a Diagnostic 452-Base-Pair DNA Fragment Discriminates between Cryptosporidium parvum and C. meleagridis and between C. parvum Isolates of Human and Animal Origin

2002 ◽  
Vol 68 (4) ◽  
pp. 2071-2076 ◽  
Author(s):  
K. Guyot ◽  
A. Follet-Dumoulin ◽  
C. Recourt ◽  
E. Lelièvre ◽  
J. C. Cailliez ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Cryptosporidium Parvum ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Animal Origin ◽  
Polymorphism Analysis ◽  
Diagnostic Fragment ◽  
Restriction Fragment Length

ABSTRACT Genomic DNAs from human Cryptosporidium isolates previously typed by analysis of the 18S ribosomal DNA locus (Cryptosporidium parvum bovine genotype, C. parvum human genotype, Cryptosporidium meleagridis, and Cryptosporidium felis) were used to amplify the diagnostic fragment described by Laxer et al. (M. A. Laxer, B. K. Timblin, and R. J. Patel, Am. J. Trop. Med. Hyg., 45:688-694, 1991). The obtained 452-bp amplified fragments were sequenced and aligned with the homologous Cryptosporidium wrairi sequence. Polymorphism was exploited to develop a restriction fragment length polymorphism method able to discriminate Cryptosporidium species and C. parvum genotypes.

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