PCR and Real Time PCR for the Detection of Cryptosporidium parvum Oocyst DNA

2011 ◽  
Vol 59 (3) ◽  
pp. 115-120 ◽  
Keyword(s):  
Real Time ◽  
Cryptosporidium Parvum ◽  
Real Time Pcr ◽  
Cryptosporidium Parvum Oocyst

Quantification of Cryptosporidium parvum in anaerobic digesters treating manure by (reverse-transcription) quantitative real-time PCR, infectivity and excystation tests

2006 ◽  
Vol 53 (8) ◽  
pp. 195-202 ◽  
Author(s):  
G. Garcés ◽  
M. Effenberger ◽  
M. Najdrowski ◽  
C. Wackwitz ◽  
A. Gronauer ◽  
...  
Keyword(s):  
Anaerobic Digestion ◽  
Real Time ◽  
Cryptosporidium Parvum ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
Combined Treatment ◽  
Quantitative Real Time Pcr ◽  
Anaerobic Digesters ◽  
Hsp70 Mrna ◽  
Cryptosporidium Parvum Oocysts

The survival of Cryptosporidium parvum oocysts in anaerobic digesters treating manure was investigated for mesophilic, thermophilic, and a combined treatment (mesophilic–thermophilic–mesophilic) under different retention times of oocysts in the reactors. C. parvum DNA was extracted with an optimised protocol, and its amount determined by quantitative real-time PCR (qPCR). Results indicated noteworthy differences in DNA content after the different treatments. DNA was not degraded during the process. However, excystation and infectivity tests showed a reduction of viable oocyst numbers of ≥2 and ≥5 log units after the thermophilic treatment in two different experiments. Thus qPCR-targeting DNA can overestimate the number of oocysts that survive and remain viable after anaerobic digestion. However, targeting DNA is suitable to indicate the presence or absence of oocysts. Reverse transcription qPCR (RT-qPCR) targeting C. parvum hsp70 mRNA successfully indicated the presence of viable fraction of oocysts.

Detecting Cryptosporidium parvum and Giardia lamblia by coagulation concentration and real-time PCR quantification

2012 ◽  
Vol 7 (1) ◽  
pp. 49-54 ◽  
Author(s):  
Huining Zhang ◽  
Xiaohu Zhang ◽  
Shuting Zhang ◽  
Bo Wei ◽  
Qipei Jiang ◽  
...  
Keyword(s):  
Real Time ◽  
Cryptosporidium Parvum ◽  
Real Time Pcr ◽  
Giardia Lamblia

Evaluation of alternative DNA extraction processes and real-time PCR for detecting Cryptosporidium parvum in drinking water

2015 ◽  
Vol 15 (6) ◽  
pp. 1295-1303
Author(s):  
Gina H. Kimble ◽  
Vincent R. Hill ◽  
James E. Amburgey
Keyword(s):  
Drinking Water ◽  
Real Time ◽  
Dna Extraction ◽  
Cryptosporidium Parvum ◽  
Water Samples ◽  
Real Time Pcr ◽  
Extraction Procedure ◽  
The United States ◽  
Detection Sensitivity ◽  
Spin Column

USEPA Method 1623 is the standard method in the United States for the detection of Cryptosporidium in water samples, but quantitative real-time polymerase chain reaction (qPCR) is an alternative technique that has been successfully used to detect Cryptosporidium in aqueous matrices. This study examined various modifications to a commercial nucleic acid extraction procedure in order to enhance PCR detection sensitivity for Cryptosporidium. An alternative DNA extraction buffer allowed for qPCR detection at lower seed levels than a commercial extraction kit buffer. In addition, the use of a second spin column cycle produced significantly better detection (P = 0.031), and the volume of Tris–EDTA buffer significantly affected crossing threshold values (P = 0.001). The improved extraction procedure was evaluated using 10 L of tap water samples processed by ultrafiltration, centrifugation and immunomagnetic separation. Mean recovery for the sample processing method was determined to be 41% using microscopy and 49% by real-time PCR (P = 0.013). The results of this study demonstrate that real-time PCR can be an effective alternative for detecting and quantifying Cryptosporidium parvum in drinking water samples.

scholarly journals Detection and Genotyping of Oocysts of Cryptosporidium parvum by Real-Time PCR and Melting Curve Analysis

2002 ◽  
Vol 40 (9) ◽  
pp. 3237-3244 ◽  
Author(s):  
S. Tanriverdi ◽  
A. Tanyeli ◽  
F. Baslamisli ◽  
F. Koksal ◽  
Y. Kilinc ◽  
...  
Keyword(s):  
Real Time ◽  
Cryptosporidium Parvum ◽  
Real Time Pcr ◽  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis

scholarly journals Detection and differentiation of Cryptosporidium hominis and Cryptosporidium parvum by dual TaqMan assays

2008 ◽  
Vol 57 (9) ◽  
pp. 1099-1105 ◽  
Author(s):  
N. Jothikumar ◽  
A. J. da Silva ◽  
I. Moura ◽  
Y. Qvarnstrom ◽  
V. R. Hill
Keyword(s):  
Real Time ◽  
Cryptosporidium Parvum ◽  
Real Time Pcr ◽  
18S Rrna ◽  
Laboratory Diagnosis ◽  
Taqman Assay ◽  
Cryptosporidium Hominis ◽  
Specific Assay ◽  
Outbreak Investigations ◽  
Stool Specimens

Rapid identification of the two major species of Cryptosporidium associated with human infections, Cryptosporidium hominis and Cryptosporidium parvum, is important for investigating outbreaks of cryptosporidiosis. This study reports the development and validation of a real-time PCR TaqMan procedure for detection of Cryptosporidium species and identification of C. hominis and C. parvum in stool specimens. This procedure comprised a generic TaqMan assay targeting the 18S rRNA for sensitive detection of Cryptosporidium species, as well as two other TaqMan assays for identification of C. hominis and C. parvum. The generic Cryptosporidium species assay can be duplexed with the C. parvum-specific assay. The generic Cryptosporidium species assay was able to detect ten Cryptosporidium species and did not cross-react with a panel of ten other protozoan parasites. The generic Cryptosporidium species assay could detect 1–10 oocysts in a 300 μl stool specimen, whilst each of the species-specific TaqMan assays had detection sensitivities that were approximately tenfold higher. The 18S rRNA assay was found to detect Cryptosporidium species in 49/55 DNA extracts from stool specimens containing either C. hominis or C. parvum. The C. hominis TaqMan assay correctly identified C. hominis in 24/31 validation panel specimens containing this species. The C. parvum-specific assay correctly identified C. parvum in 21/24 validation panel specimens containing this species. This real-time PCR procedure was used to detect and identify C. hominis and C. parvum in stool specimens from outbreak investigations in the USA and Botswana, resulting in identification of C. hominis and/or C. parvum in 66/67 stool specimens shown to be positive for these species using other techniques. From the outbreak specimens tested, the TaqMan procedure was found to have a specificity of 94 %. This TaqMan PCR procedure should be a valuable tool for the laboratory diagnosis of cryptosporidiosis caused by C. hominis and C. parvum during outbreak investigations.

scholarly journals Multicenter Comparative Study of Six Cryptosporidium parvum DNA Extraction Protocols Including Mechanical Pretreatment from Stool Samples

2020 ◽  
Vol 8 (9) ◽  
pp. 1450
Author(s):  
Nicolas Valeix ◽  
Damien Costa ◽  
Louise Basmaciyan ◽  
Stéphane Valot ◽  
Anne Vincent ◽  
...  
Keyword(s):  
Comparative Study ◽  
Real Time ◽  
Dna Extraction ◽  
Cryptosporidium Parvum ◽  
Real Time Pcr ◽  
Sample Pretreatment ◽  
Dna Amplification ◽  
Extraction Methods ◽  
Mechanical Pretreatment ◽  
Stool Samples

Background: Nowadays, many commercial kits allow the detection of Cryptosporidium sp. in stool samples after deoxyribonucleic acid (DNA) extraction. Protocols of stool pretreatment have been proposed to optimize oocysts’ DNA extraction. Among them, mechanical grinding was reported to improve the performance of Cryptosporidium oocysts’ DNA extraction. Methods: A multicenter comparative study was conducted within the framework of the French National Reference Center-Expert Laboratory for Cryptosporidiosis. Six extraction systems (i.e., manual or automated) associated with various mechanical pretreatment protocols, were compared for the Cryptosporidium parvum oocyst’ DNA extraction, before amplification using the same real-time PCR method targeting. Results: The sensitivity of real-time PCR assay was unequally impacted by the pretreatment/extraction protocol. We observed significant differences for the lowest concentrations of C. parvum oocysts (i.e., 0–94.4% and 33.3–100% respectively for 10 and 50 oocysts/mL). All in all, the protocol using Quick DNA Fecal/Soil Microbe-Miniprep® manual kit showed the best performances. In addition, optimal performances of mechanical pretreatment were obtained by combining a grinding duration of 60 s with a speed of 4 m/s using Fastprep24® with Lysing Matrix E®. Conclusions: Sample pretreatment, as well as the extraction method, needs to be properly adapted to improve the diagnostic performances of the C. parvum DNA amplification methods.

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