scholarly journals Real-time PCR detection and speciation of Cryptosporidium infection using Scorpion probes

2006 ◽  
Vol 55 (9) ◽  
pp. 1217-1222 ◽  
Author(s):  
Suzanne E. Stroup ◽  
Shantanu Roy ◽  
John Mchele ◽  
Venance Maro ◽  
Simon Ntabaguzi ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rflp Analysis ◽  
18S Rrna Gene ◽  
Human Infection ◽  
Rrna Gene ◽  
Dna Extraction Method ◽  
Stool Samples ◽  
Stool Specimens ◽  
Human Stool

At least eight species of Cryptosporidium can cause human infection and disease. A real-time PCR (qPCR) assay based on the 18S rRNA gene and utilizing a Scorpion probe was developed to detect all human-pathogenic Cryptosporidium without the usual need for nested amplification. Sensitivity of detection in stool samples was highest using a glass bead-based DNA extraction method (under 103 oocysts per stool sample). The assay was validated against 123 human stool specimens from Bangladesh and Tanzania, exhibited a sensitivity and specificity of >91 % versus microscopy, and detected an additional eight microscopy-negative infections. Cryptosporidium parvum-specific and Cryptosporidium meleagridis-specific Scorpion qPCR assays that provided 100 % accurate speciation compared with VspI RFLP analysis and sequencing were developed subsequently. These Scorpion probe qPCR assays are simpler to perform than existing nested PCR and RFLP methods for diagnosis and epidemiological investigation of cryptosporidiosis.

scholarly journals  Recovery of Cryptosporidium from spiked water and stool samples measured by PCR and real time PCR

2012 ◽  
Vol 57 (No. 5) ◽  
pp. 224-232 ◽  
Author(s):  
M. Adamska ◽  
A. Leonska-Duniec ◽  
M. Sawczuk ◽  
A. Maciejewska ◽  
B. Skotarczak
Keyword(s):  
Real Time ◽  
Water Samples ◽  
Real Time Pcr ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
High Concentration ◽  
Intestinal Protozoan ◽  
Wide Range ◽  
Stool Samples

Cryptosporidium parvum is a common intestinal protozoan parasite infecting humans and a wide range of animals, whose diagnostics present considerable difficulties. These arise from the exceptionally robust nature of the oocyst’s walls, which necessitates more stringent treatments for disruption and recovery of DNA for analysis using molecular methods. In the case of water, which is the major source of Cryptosporidium oocysts, investigations concern the detection of the presence of the oocysts. Their concentration in water is very low, and moreover, many substances that may have significance as inhibitors of DNA amplification, are present in environmental water and stool. We have carried out trials in order to assess the effectiveness of recovery of C. parvum oocysts, from spiked environmental and distilled water samples, filtrated and concentrated with the use of special laboratory equipment. Inactivation of inhibitors was carried out with use of bovine serum albumin (BSA) in PCR mixes at ten different concentrations. DNA extraction was carried out from stool samples spiked with C. parvum oocysts, concentrated using two methods, and unconcentrated. Nested PCR and a TaqMan nested real time PCR assay, targeting the 18S rRNA gene, was used to detect C. parvum DNA in spiked water and additionally in spiked stool samples. The obtained results showed that losses of C. parvum oocysts occur during the filtration and concentration of spiked water samples. The addition of small amounts of BSA (5–20 ng/µl) to PCR and TaqMan PCR mixes increases the sensitivity of both methods, but a high concentration of BSA (100 ng/µl and above) has an inhibiting effect on the polymerase reaction. The extraction of DNA from C. parvum oocysts from spiked stool samples preceded by concentration with PBS, ether and Percoll resulted in a higher copy number of the 18S rRNA gene.  

scholarly journals Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Bead Beating ◽  
Stool Samples ◽  
Pcr Assays ◽  
Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

scholarly journals Genotyping of Acantamoeba spp. from rhisophere in Hungary

2020 ◽  
Vol 67 (3) ◽  
pp. 171-175
Author(s):  
Erika Orosz ◽  
Katalin Posta
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Human Pathogens ◽  
Quantitative Real Time Pcr ◽  
Potential Health ◽  
Granulomatous Amoebic Encephalitis ◽  
Related Genotype ◽  
Biological Methods

The protista Acanthamoeba is a free-living amoeba existing in various environments. A number of species among protista are recognized as human pathogens, potentially causing Acanthamoeba keratitis (AK), granulomatous amoebic encephalitis (GAE), and chronic granulomatous lesions. In this study, 10 rhizosphere samples were collected from maize and alfalfa plants in experimental station at Institute of Genetics, Microbiology and Biotechnology, Szent István University. We detected Acanthamoeba based on the quantitative real-time PCR assay and sequence analysis of the 18S rRNA gene. All studied molecular biological methods are suitable for the detection of Acanthamoeba infection in humans. The quantitative real-time PCR-based methods are more sensitive, simple, and easy to perform; moreover, these are opening avenue to detect the effect of number of parasites on human disease. Acanthamoeba species were detected in five (5/10; 50%) samples. All Acanthamoeba strains belonged to T4 genotype, the main AK-related genotype worldwide. Our result confirmed Acanthamoeba strains in rhizosphere that should be considered as a potential health risk associated with human activities in the environment.

Development of a TaqMan based real-time PCR assay for detection of Clonorchis sinensis DNA in human stool samples and fishes

2012 ◽  
Vol 61 (1) ◽  
pp. 183-186 ◽  
Author(s):  
Xian-Quan Cai ◽  
Hai-Qiong Yu ◽  
Jian-Shan Bai ◽  
Jian-Dong Tang ◽  
Xu-Chu Hu ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Clonorchis Sinensis ◽  
Pcr Assay ◽  
Stool Samples ◽  
Human Stool

scholarly journals Detection of Arcobacter Species in Human Stool Samples by Culture and Real-time PCR

2020 ◽  
Vol 66 (5) ◽  
pp. 431-438
Author(s):  
YUKO YAMAUCHI ◽  
YUKI UEHARA ◽  
SÉBASTIEN BOUTIN ◽  
NORIO YAMAMOTO ◽  
KYOKO KUWAHARA-ARAI ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Stool Samples ◽  
Human Stool

Improving the specific diagnosis of trematode, cestode and nematode infections by a multiplex single-tube real-time PCR assay

2019 ◽  
Vol 72 (7) ◽  
pp. 487-492 ◽  
Author(s):  
Samson S Y Wong ◽  
Rosana W S Poon ◽  
Kelvin K W To ◽  
Jasper F W Chan ◽  
Gang Lu ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Molecular Diagnostic ◽  
Rrna Gene ◽  
28S Rrna ◽  
Pcr Assay ◽  
Single Tube ◽  
Histological Sections ◽  
Pcr Products ◽  
Stool Samples

AimsHelminth infections are becoming uncommon in high-income countries and laboratory staff may lose expertise in their morphological identification, especially in histological sections where speciation of helminths is challenging. Commercially available molecular diagnostic panels for faecal specimens only offer tests for protozoa but not helminths. We aim to improve the identification accuracy of helminths using a multiplex PCR assay.MethodsWe designed three pairs of PCR primers and probes targeting multicopy genes for a multiplex single-tube real-time PCR assay which covers 16 trematode (28S rRNA gene), 24 cestode (cox1 gene) and 33 nematode (cox1 gene) species. Helminths (n=27) from faecal samples (n=10), fresh parasites (n=11), formalin-fixed specimens (n=4), cerebrospinal fluid (n=1) and bile (n=1) were examined morphologically and tested by PCR. Fifty stool samples negative for parasites by microscopy were also tested.ResultsThe PCR assay correctly identified the genera of all tested helminths. Agarose gel electrophoresis and sequencing of the purified PCR amplicons confirmed that the PCR products were of correct sizes with 100% correlation with the respective species. Sequencing of the cox1 gene failed to identify Capillaria spp. in one sample owing to the lack of corresponding sequences in GenBank. PCR and sequencing of the nematode 18S rRNA gene using consensus primers showed 100% homology with Capillaria spp. sequence. No positive PCR products were found in the negative stool samples.ConclusionsThe highly specific test correctly identified all helminths in our cohort. It is a useful adjunct to helminth identification in difficult situations such as histological sections.

scholarly journals Detection of Four Plasmodium Species in Blood from Humans by 18S rRNA Gene Subunit-Based and Species-Specific Real-Time PCR Assays

2004 ◽  
Vol 42 (12) ◽  
pp. 5636-5643 ◽  
Author(s):  
M. Rougemont ◽  
M. Van Saanen ◽  
R. Sahli ◽  
H. P. Hinrikson ◽  
J. Bille ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
18S Rrna ◽  
Plasmodium Species ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Pcr Assays ◽  
Species Specific

scholarly journals Development and Validation of a Real-Time PCR Method To Quantify Rumen Protozoa and Examination of Variability between Entodinium Populations in Sheep Offered a Hay-Based Diet

2006 ◽  
Vol 72 (1) ◽  
pp. 200-206 ◽  
Author(s):  
Lucy C. Skillman ◽  
Andrew F. Toovey ◽  
Andrew J. Williams ◽  
André-Denis G. Wright
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Advantages And Disadvantages ◽  
Pcr Efficiency ◽  
Rumen Protozoa ◽  
Pcr Method ◽  
Serial Dilutions

ABSTRACT PCR and real-time PCR primers for the 18S rRNA gene of rumen protozoa (Entodinium and Dasytricha spp.) were designed, and their specificities were tested against a range of rumen microbes and protozoal groups. External standards were prepared from DNA extracts of a rumen matrix containing known numbers and species of protozoa. The efficiency of PCR (ε) was calculated following amplification of serial dilutions of each standard and was used to calculate the numbers of protozoa in each sample collected; serial dilutions of DNA were used similarly to calculate PCR efficiency. Species of Entodinium, the most prevalent of the rumen protozoa, were enumerated in rumen samples collected from 100 1-year-old merino wethers by microscopy and real-time PCR. Both the counts developed by the real-time PCR method and microscopic counts were accurate and repeatable, with a strong correlation between them (R 2 = 0.8), particularly when the PCR efficiency was close to optimal (i.e., two copies per cycle). The advantages and disadvantages of each procedure are discussed. Entodinium represented on average 98% of the total protozoa, and populations within the same sheep were relatively stable, but greater variation occurred between different sheep (100 and 106 entodinia per gram of rumen contents). With this inherent variability, it was estimated that, to detect a statistically significant (P = 0.05) 20% change in Entodinium populations, 52 sheep per treatment group would be required.

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