scholarly journals Development of Molecular Diagnosis Using Multiplex Real-Time PCR and T4 Phage Internal Control to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis from Human Stool Samples

2018 ◽  
Vol 56 (5) ◽  
pp. 419-427 ◽  
Author(s):  
Ji-Hun Shin ◽  
Sang-Eun Lee ◽  
Tong Soo Kim ◽  
Da-Won Ma ◽  
Shin-Hyeong Cho ◽  
...  
Keyword(s):  
Real Time ◽  
Molecular Diagnosis ◽  
Cryptosporidium Parvum ◽  
Internal Control ◽  
Real Time Pcr ◽  
Giardia Lamblia ◽  
Cyclospora Cayetanensis ◽  
T4 Phage ◽  
Stool Samples ◽  
Human Stool

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 Genotype-specific detection of Giardia lamblia in stool samples of diarrhoeal and non-diarrhoeal patients in Dhaka, Bangladesh

1970 ◽  
Vol 20 (2) ◽  
pp. 183-189 ◽  
Author(s):  
Md Masud Alam ◽  
Mohammad Ilias ◽  
Md Abdullah Siddique ◽  
Md Mamun Kabir ◽  
Farida Nazib ◽  
...  
Keyword(s):  
Positive Result ◽  
Real Time ◽  
Real Time Pcr ◽  
Giardia Lamblia ◽  
Specific Antigen ◽  
Diarrhoeal Disease ◽  
Giardia Cyst ◽  
Positive Stool ◽  
Stool Samples ◽  
Assemblage B

Two major genotypic assemblages (A and B) of Giardia lamblia infect humans. A single-vessel multiplex real-time PCR assay was used that genotypes Giardia infections into assemblages A and/or B directly from fecal samples. In this study, 157 diarrhoeal (symptomatic) and non-diarrhoeal (asymptomatic) stool samples collected from the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) and Bangabandhu Sheikh Mujib Medical University (BSMMU) hospital, respectively were analyzed to determine whether an association exists between infections with G. lamblia assemblages A or B and diarrhea in Bangladesh. Of the 157 stool samples, Giardia cysts were observed in 35 by microscopy and 127 showed positive result for Giardia cyst specific antigen. The 127 ELISA positive samples were assayed for genotyping by real?time polymerase chain reaction. Of the 117 real-time PCR positive stool samples, 15 were positive for G. lamblia assemblage A, 96 were positive for assemblage B and 6 samples showed positive result for both G. lamblia assemblage A and B infections. Higher ratios for diarrhea were observed for assemblage A infections, whereas higher parasite DNA loads and a higher overall rate were observed for assemblage B infections in both diarrhoeal and non-diarrhoeal patients. Real-time PCR is, therefore, useful as an additional test supplementary to microscopy or enzyme immunoassay to detect genotypes of Giardia. Key words: Giardia lamblia; Genotypes; Multiplex real-time PCR; Immunoassay DOI: http://dx.doi.org/10.3329/dujbs.v20i2.8979 DUJBS 2011; 20(2): 183-189

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

Routine use of duplex real-time PCR assays including a commercial internal control for molecular diagnosis of opportunistic DNA virus infections

2012 ◽  
Vol 185 (1) ◽  
pp. 136-141 ◽  
Author(s):  
Sonia Burrel ◽  
Christelle Fovet ◽  
Christel Brunet ◽  
Lydia Ovaguimian ◽  
Nathalie Hamm ◽  
...  
Keyword(s):  
Real Time ◽  
Molecular Diagnosis ◽  
Internal Control ◽  
Real Time Pcr ◽  
Virus Infections ◽  
Dna Virus ◽  
Pcr Assays

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

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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