scholarly journals A New Duplex PCR-Assay for the Detection and Identification of Paracoccidioides Species

2021 ◽  
Vol 7 (3) ◽  
pp. 169
Author(s):  
Breno Gonçalves Pinheiro ◽  
Ana Paula Pôssa ◽  
Paula Portella Della Terra ◽  
Jamile Ambrósio de Carvalho ◽  
Giannina Ricci ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Paracoccidioides Brasiliensis ◽  
Cost Effective ◽  
Duplex Pcr ◽  
Pcr Assay ◽  
Accurate Identification ◽  
Exon 2 ◽  
Detection And Identification ◽  
Species Specific ◽  
Duplex Pcr Assay

Paracoccidioidomycosis (PCM) is a life-threatening systemic fungal infection caused by members of the Paracoccidioides brasiliensis complex and P. lutzii. Routine diagnoses of PCM down to the species level using classical mycological approaches are unspecific due to overlapping phenotypes. There is an urgent need for specific, sensitive, and cost-effective molecular tools to diagnose PCM. Variation among the exon-2 of the gp43 gene was exploited to design species-specific primer pairs to discriminate between members of the P. brasiliensis complex and P. lutzii in a duplex PCR assay. Primer-BLAST searches revealed highly species-specific primers, and no significant region of homology was found against DNA databases except for Paracoccidioides species. Primers PbraCx-F and PbraCx-R targeting P. brasiliensis DNA produced an amplicon of 308 bp, while primers Plu-F and Plu-R targeting P. lutzii DNA generated an amplicon of 142 bp. The lower limit of detection for our duplex PCR assay was 1 pg of gDNA. A panel of 62 Paracoccidioides revealed 100% specificity (AUC = 1.000, 95%CI 0.972–1.000, p < 0.0001) without cross-reacting with other medically relevant fungi or human DNA. As a proof of concept, we demonstrated the accurate identification of the P. brasiliensis complex (n = 7) or P. lutzii (n = 6) from a broad range of formalin-fixed, paraffin-embedded (FFPE) tissues of PCM patient’s organs. In four cases, FFPE PCR results confirmed, for the first time, co-infection due to P. brasiliensis (S1) and P. lutzii in the same biopsy. Our duplex PCR assay is useful to detect and differentiate members of the P. brasiliensis complex and P. lutzii, providing clinical laboratories with an important tool to be applied routinely, especially in atypical cases such as those featuring negative serology and positive mycological examination of clinical specimens as well as for the investigation of putative co-infection cases. This will likely benefit thousands of infected patients every year in a wide area of the Americas.

scholarly journals Detection of Trypanosoma cruzi and Trypanosoma rangeli Infection by Duplex PCR Assay Based on Telomeric Sequences

2003 ◽  
Vol 10 (5) ◽  
pp. 775-779 ◽  
Author(s):  
Miguel Angel Chiurillo ◽  
Gladys Crisante ◽  
Agustina Rojas ◽  
Andreina Peralta ◽  
Manuel Dias ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Human Subjects ◽  
Simultaneous Detection ◽  
Duplex Pcr ◽  
Trypanosoma Rangeli ◽  
Pcr Assay ◽  
Telomeric Sequences ◽  
Species Specific ◽  
Duplex Pcr Assay ◽  
Reduviid Bugs

ABSTRACT We used the species specificity and repetitious nature of subtelomeric kinetoplastida sequences to generate a duplex PCR assay for the simultaneous detection of Trypanosoma cruzi and Trypanosoma rangeli in experimentally and naturally infected triatomine (Reduviid) bugs and in infected human subjects. The assay was species specific and was capable of detecting 1/20th of T. cruzi and 1/4th of T. rangeli cell equivalents without complementary hybridization. In addition, the PCR-based assay was robust enough for direct application to difficult biological samples such as Reduviid feces or guts and was capable of recognizing all T. cruzi and T. rangeli strains and lineages. Because the assay primers amplify entirely different target sequences, no reaction interference was observed, facilitating future adaptation of this assay to an automated format.

scholarly journals Conversion of sequence-characterized amplified region (SCAR) bands into high-throughput DNA markers based on RAPD technique for detection of the stem nematode Ditylenchus dipsaci in crucial plant hosts

2008 ◽  
Vol 53 (No. 3) ◽  
pp. 97-104 ◽  
Author(s):  
M. Zouhar ◽  
M. Marek ◽  
O. Douda ◽  
J. Mazáková ◽  
P. Ryšánek
Keyword(s):  
Cost Effective ◽  
Healthy Plant ◽  
Host Preferences ◽  
Sequence Characterized Amplified Region ◽  
Detection And Identification ◽  
Plant Hosts ◽  
Cost Effective Technique ◽  
Ditylenchus Dipsaci ◽  
Species Specific ◽  
Template Dna

<i>Ditylenchus dipsaci</i>, the stem nematode, is a migratory endoparasite of over 500 species of angiosperms. The main method of <i>D. dipsaci</i> control is crop rotation, but the presence of morphologically indistinguishable host races with different host preferences makes rotation generally ineffective. Therefore, a sensitive, rapid, reliable, as well as cost effective technique is needed for identification of <i>D. dipsaci</i> in biological samples. This study describes the development of species-specific pairs of PCR oligonucleotides for detection and identification of the <i>D. dipsaci</i> stem nematode in various plant hosts. Designed DIT-2 primer pair specifically amplified a fragment of 325 bp, while DIT-5 primer pair always produced a fragment of 245 bp in all <i>D. dipsaci</i> isolates. Two developed SCAR primer pairs were further tested using template DNA extracted from a collection of twelve healthy plant hosts; no amplification was however observed. The developed PCR protocol has proved to be quite sensitive and able to specifically detect <i>D. dipsaci</i> in artificially infested plant tissues.

scholarly journals Duplex-PCR assay for the detection of adenovirus and respiratory syncytial virus in nasopharyngeal samples

2009 ◽  
Vol 104 (1) ◽  
pp. 118-120 ◽  
Author(s):  
Juliana Cristina Marinheiro ◽  
Roberta Braga Sanalios ◽  
Daniela Carvalho dos Santos ◽  
Cristovão Alves da Costa ◽  
Charlotte Marianna Hársi
Keyword(s):  
Respiratory Syncytial Virus ◽  
Duplex Pcr ◽  
Pcr Assay ◽  
Syncytial Virus ◽  
Duplex Pcr Assay

Rapid detection of Yersinia enterocolitica serotype O:3 using a duplex PCR assay

2018 ◽  
Vol 154 ◽  
pp. 107-111 ◽  
Author(s):  
Leonardo Alves Rusak ◽  
Rodrigo de Castro Lisboa Pereira ◽  
Isabelle Geoffroy Freitag ◽  
Cristina Barroso Hofer ◽  
Ernesto Hofer ◽  
...  
Keyword(s):  
Yersinia Enterocolitica ◽  
Rapid Detection ◽  
Duplex Pcr ◽  
Pcr Assay ◽  
Serotype O ◽  
Duplex Pcr Assay

A duplex PCR assay for authentication of Ocimum basilicum L. and Ocimum tenuiflorum L in Tulsi churna

Food Control ◽  
2021 ◽  
pp. 108790
Author(s):  
Tasnim Travadi ◽  
Sonal Sharma ◽  
Ramesh Pandit ◽  
Mital Nakrani ◽  
Chaitanya Joshi ◽  
...  
Keyword(s):  
Ocimum Basilicum ◽  
Duplex Pcr ◽  
Pcr Assay ◽  
Ocimum Tenuiflorum ◽  
Duplex Pcr Assay

scholarly journals Species-Specific Identification of Human Adenoviruses by a Multiplex PCR Assay

2000 ◽  
Vol 38 (11) ◽  
pp. 4114-4120 ◽  
Author(s):  
WanHong Xu ◽  
Mike C. McDonough ◽  
Dean D. Erdman
Keyword(s):  
Multiplex Pcr ◽  
Human Adenovirus ◽  
Cost Effective ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Amplicon Size ◽  
Species Specific ◽  
Fiber Gene ◽  
Amplification Reaction

A multiplex PCR assay was developed by using primers to the fiber gene that could differentiate human adenovirus (Ad) species A through F in a single amplification reaction. The assay correctly identified the species of all 49 recognized Ad prototype strains as well as 180 geographically and temporally diverse Ad field isolates. Ad serotype 6 (Ad6) (species C), Ad16 (species B), Ad31 (species A), and Ad40 and Ad41 (species F) could also be distinguished by amplicon size within each respective species. In comparison, a previously described Ad species-specific multiplex PCR assay that used primers to the Ad hexon gene gave equivocal results with several serotypes of species B, whereas our multiplex assay amplified all species B serotypes equally well. Our multiplex PCR assay will permit rapid, accurate, and cost-effective classification of Ad isolates.

scholarly journals Development and characterization of a duplex PCR assay in Chinese sturgeon (Acipenser sinensis) for genetic analysis

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yacheng Hu ◽  
Xueqing Liu ◽  
Jing Yang ◽  
Kan Xiao ◽  
Binzhong Wang ◽  
...  
Keyword(s):  
Genetic Analysis ◽  
Duplex Pcr ◽  
Chinese Sturgeon ◽  
Pcr Assay ◽  
Acipenser Sinensis ◽  
Duplex Pcr Assay
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