LNA probes in a real-time TaqMan PCR assay for genotyping of<i>Giardia duodenalis</i>in wastewaters

Alonso, J.L.; Amorós, Inmaculada; Cuesta, G.

doi:10.1111/j.1365-2672.2009.04559.x

Cited by 11 publications

(9 citation statements)

References 25 publications

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“…The assessed real-time screening PCRs for G. duodenalis targeted the 18S rRNA gene [ 26 ], the gdh (glutamate dehydrogenase) gene [ 27 ], and the bg (beta-giardin) gene [ 25 ], respectively. The compared duplex real-time PCRs for the discrimination of the G. duodenalis assemblages A and B targeted the bg gene excluding [ 25 ] and including [ 28 ] the use of LNA (locked nucleic acid)–containing probes as well as the tpi (triose phosphate isomerase) gene [ 29 ], respectively. Real-time PCR for Phocid herpes virus (PhHV) DNA was applied as an internal control as described [ 48 ].…”

Section: Methodsmentioning

confidence: 99%

“…Various DNA sequence targets have been tested and applied for real-time PCR-based screening for G. duodenalis as well as for diagnostic discrimination of the etiologically relevant assemblages A and B in both validation studies and epidemiological assessments, including genes encoding beta-giardin ( bg ), triose phosphate isomerase ( tpi ), 18S rRNA (=the small sub-unit of ribosomal RNA), and glutamate dehydrogenase ( gdh ) [ 19 , 20 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. In this study, three previously described real-time PCR screening assays for G. duodenalis targeting the 18S rRNA gene, the bg gene and the gdh gene, respectively [ 25 , 26 , 27 ], as well as three described real-time PCR differentiation assays targeting the bg gene ( n = 2 assays) and the tpi gene [ 25 , 28 , 29 ] were compared in head-to-head test comparisons without a reference standard applying latent class analysis [ 16 , 37 ]. Our overarching objective was to assess the influence of the choice of different target genes on the diagnostic accuracy of real-time PCR for G. duodenalis and its assemblages A and B.…”

Section: Introductionmentioning

confidence: 99%

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Comparative Evaluation of Real-Time Screening PCR Assays for Giardia duodenalis and of Assays Discriminating the Assemblages A and B

Weinreich

Hahn²,

Eberhardt

et al. 2022

Microorganisms

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Due to superior sensitivity compared to traditional microscopy, real-time PCR has been well established for the diagnosis of Giardia duodenalis in human stool samples. In this study, screening real-time PCRs for different target genes of G. duodenalis, i.e., the 18S rRNA gene, the gdh (glutamate dehydrogenase) gene and the bg (beta-giardin) gene, were comparatively assessed next to various real-time PCR assays for the discrimination of the assemblages A and B of G. duodenalis targeting the bg gene with and without locked nucleic acid–containing probes as well as the tpi (triose phosphate isomerase) gene. The screening PCRs were assessed by including 872 non-preselected samples with a high pre-test probability for G. duodenalis in the statistical analysis, while 53 G. duodenalis-positive samples as indicated by at least two screening PCRs were finally included in the assessment of the assemblage-specific PCRs. For the screening PCRs, sensitivity estimated with latent class analysis (LCA) ranged from 17.5% to 100%, specificity from 92.3% to 100% with an accuracy-adjusted prevalence of 7.2% for G. duodenalis within the non-preselected sample collection. In detail, sensitivity and specificity were 100% and 100% for the 18S rRNA gene-specific assay, 17.5% and 92.3% for the gdh gene-specific assay, and 31.7% and 100% for the bg gene-specific assay, respectively. Agreement kappa was slight with only 15.5%. For the assemblage-specific PCRs, estimated sensitivity ranged from 82.1% to 100%, specificity from 84.0% to 100% with nearly perfect agreement kappa of 90.1% for assemblage A and yet substantial agreement of 74.8% for assemblage B. In detail for assemblage A, sensitivity and specificity were 100% and 100% for the bg gene-specific assay without locked nucleic acids (LNA) as well as 100% and 97.8% for both the bg gene-specific assay with LNA and the tri gene-specific assay, respectively. For assemblage B, sensitivity and specificity were 100% and 100% for the bg gene-specific assay without LNA, 96.4% and 84.0% for the bg gene-specific assay with LNA, and 82.1% and 100% for the tri gene-specific assay, respectively. Within the assessed sample collection, the observed proportion comprised 15.1% G. duodenalis assemblage A, 52.8% G. duodenalis assemblage B and 32.1% non-resolved assemblages. Only little differences were observed regarding the cycle threshold (Ct) values when comparing the assays. In conclusion, best diagnostic accuracy was shown for an 18S rRNA gene-specific screening assay for G. duodenalis and for a differentiation assay discriminating the G. duodenalis assemblages A and B by targeting the bg gene with probes not containing locked nucleic acids. By adding additional highly specific competitor assays for confirmation testing, diagnostic specificity can be further increased on the cost of sensitivity if optimized specificity is desired.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Comparative Evaluation of Real-Time Screening PCR Assays for Giardia duodenalis and of Assays Discriminating the Assemblages A and B

Weinreich

Hahn²,

Eberhardt

et al. 2022

Microorganisms

View full text Add to dashboard Cite

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“…Locked nucleic acids obey the Watson-Crick pairing rules, but have an increased specificity and high affinity to complementary DNA (Sanjay et al 1998;Vester and Wengel 2004;Koshkin et al 1998;Nolan et al 2006). Due to such advantages, LNA or LNA/DNA mixmers (hereinafter called LNA) have been used for real-time PCR (Reynisson et al 2006;Alonso et al 2010) and fluorescent in situ hybridization (Silahtaroglu et al 2003;Kubota et al 2006). Thus, the purpose of the present study is the evaluation of the performance of LNA probes in a real-time PCR assay for quantification of Giardia and Cryptosporidium in sewage.…”

Section: Introductionmentioning

confidence: 99%

Development and evaluation of a real-time PCR assay for quantification of Giardia and Cryptosporidium in sewage samples

Alonso

Amorós

Cañigral

2010

Appl Microbiol Biotechnol

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Cryptosporidium and Giardia are major causes of diarrheal disease in humans worldwide and are major causes of protozoan waterborne diseases. Two DNA TaqMan PCR-based Giardia and Cryptosporidium methods targeting a 74-bp sequence of the β-giardin Giardia gene and a 151-bp sequence of the COWP Cryptosporidium gene, respectively, were used as models to compare two different LNA/DNA TaqMan probes to improve the detection limit in a real-time PCR assay. The LNA probes were the most sensitive resulting in 0.96 to 1.57 lower C t values than a DNA Giardia TaqMan probe and 0.56 to 2.21 lower than a DNA Cryptosporidium TaqMan probe. Evaluation of TaqMan Giardia and Cryptosporidium probes with LNA substitutions resulted in real-time PCR curves with an earlier C t values than conventional DNA TaqMan probes. In conclusion, the LNA probes could be useful for more sensitive detection limits.

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“…Molecular diversity indexes of Giardia duodenalis based on β-giardin locus (592 bp, n = 106). References [ 19 , 25 , 26 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 ] are cited in the Supplementary File.…”

mentioning

confidence: 99%

Genotypic and Epidemiologic Profiles of Giardia duodenalis in Four Brazilian Biogeographic Regions

Calegar

Nunes

Monteiro³

et al. 2022

Microorganisms

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Human infections with gut protozoan parasites are neglected and not targeted by specific control initiatives, leading to a knowledge gap concerning their regional diversity and epidemiology. The present study aims to explore Giardia duodenalis genetic diversity and assess the epidemiologic scenario of subclinical infections in different Brazilian biogeographic regions. Cross-sectional surveys (n = 1334 subjects) were conducted in four municipalities in order to obtain fecal samples and socioenvironmental data. Microscopy of non-diarrheal feces and nucleotide sequencing of a β-giardin gene fragment were performed. From a total of 51 samples that could be sequenced, 27 (52.9%) β-giardin sequences were characterized as assemblage A and 24 (47.1%) as assemblage B. In the Amazon, assemblage B was the most frequently detected, predominantly BIII, and with two novel sub-assemblages. Assemblage A predominated in the extra-Amazon region, with five novel sub-assemblages. Prevalence reached 17.8% (64/360) in the Amazon, 8.8% (48/544) in the Atlantic Forest, 7.4% (22/299) in Cerrado and 2.3% (3/131) in the Semiarid. People living in poverty and extreme poverty presented significantly higher positivity rates. In conclusion, subclinical giardiasis is endemic in Brazilian communities in different biogeographic regions, presenting high genetic diversity and a heterogeneous genotypic distribution.

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LNA probes in a real-time TaqMan PCR assay for genotyping ofGiardia duodenalisin wastewaters

Cited by 11 publications

References 25 publications

Comparative Evaluation of Real-Time Screening PCR Assays for Giardia duodenalis and of Assays Discriminating the Assemblages A and B

Comparative Evaluation of Real-Time Screening PCR Assays for Giardia duodenalis and of Assays Discriminating the Assemblages A and B

Development and evaluation of a real-time PCR assay for quantification of Giardia and Cryptosporidium in sewage samples

Genotypic and Epidemiologic Profiles of Giardia duodenalis in Four Brazilian Biogeographic Regions

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