Detección de Yersinia pseudotuberculosis en heces de cuyes (Cavia porcellus) utilizando una metodología microbiológica y una molecular

En este trabajo se evaluó el desempeño de dos metodologías, una microbiológica y una molecular basada en la amplificación por reacción en cadena de la polimerasa (PCR), para la detección de Yersinia pseudotuberculosis en heces de cuyes. La evaluación de cada una de las metodologías se realizó teniendo en cuenta su sensibilidad y especificidad analítica, así como su costo económico, tiempo y complejidad. La detección molecular de Y. pseudotuberculosis se realizó por PCR anidada usando iniciadores específicos para el gen de virulencia cromosomal inv, mientras que en los ensayos microbiológicos la identificación bacteriana se hizo mediante una batería comercial de perfiles bioquímicos. Se estandarizó un protocolo de amplificación en materia fecal, el cual redujo el efecto negativo que causan los inhibidores de la PCR presentes en muestras de esta naturaleza. La sensibilidad analítica más alta se observó con la metodología en la que se combinó preenriquecimiento, aislamiento microbiológico y PCR, con un rango de detección entre 1,5 x 104 y 1,5 x 103 unidades formadoras de colonias por gramo (ufc/g) de material fecal; mientras que la mayor sensibilidad obtenida en PCR anidada fue de 1,5 x 105 ufc/g de materia fecal. Tanto la metodología microbiológica como la molecular presentaron ventajas en los ensayos en los que se usó materia fecal estéril experimentalmente inoculada. Sin embargo, en muestras de materia fecal sin esterilizar la detección del microorganismo se dificultó al utilizar una única metodología, por lo que se sugiere combinar técnicas microbiológicas y moleculares para obtener un mejor desempeño diagnóstico. Yersinia pseudotuberculosis detection in feces of guinea pigs (Cavia porcellus) using microbiological and molecular methods The suitability of two methodologies, a conventional microbiological method and a molecular method, based on amplifications by Polimerase Chain Reaction (PCR) was evaluated for the detection of Yersinia pseudotuberculosis in feces of guinea pig. The analytical sensitivity and analytical specificity, as well as the economic cost, time and complexity for each method were evaluated. Molecular detection of Y. pseudotuberculosis was done by a nested PCR with specific primers for the inv chromosomal virulence gene. The microbiological confirmation was done by using a commercial identification kit. In order to reduce the side effect caused by PCR inhibitors that are normally present in feces, an amplification protocol for such type of samples was standardized. The highest sensitivity level was observed in the method that combined pre-enrichment, microbiological isolation and PCR. This method was able to detect bacterial concentrations between 1.5 x 104 and 1.5 x 103 colony-forming units per gram (CFU/g) of feces, whereas the highest diagnostic sensitivity level obtained by nested PCR was 1.5 x 105 CFU/g of feces. Both, the molecular and the microbiological methodologies, had advantages with experimentally inoculated sterile feces. Since the detection of the microorganism on samples of non-sterilized feces was difficult using either method, the use of a combination of microbiological and molecular techniques is suggested to get a better diagnostic performance for the detection of this pathogen.

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Analytical validation of a real-time hydrolysis probe PCR assay for quantifying Plasmodium falciparum parasites in experimentally infected human adults

Malaria Journal ◽

10.1186/s12936-021-03717-y ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Claire Y. T. Wang ◽

Emma L. Ballard ◽

Zuleima Pava ◽

Louise Marquart ◽

Jane Gaydon ◽

...

Keyword(s):

Clinical Trials ◽

Plasmodium Falciparum ◽

18S Rrna ◽

Drug Efficacy ◽

18S Rrna Gene ◽

Molecular Techniques ◽

Qpcr Assay ◽

Rrna Gene ◽

Analytical Sensitivity ◽

Blood Samples

Abstract Background Volunteer infection studies have become a standard model for evaluating drug efficacy against Plasmodium infections. Molecular techniques such as qPCR are used in these studies due to their ability to provide robust and accurate estimates of parasitaemia at increased sensitivity compared to microscopy. The validity and reliability of assays need to be ensured when used to evaluate the efficacy of candidate drugs in clinical trials. Methods A previously described 18S rRNA gene qPCR assay for quantifying Plasmodium falciparum in blood samples was evaluated. Assay performance characteristics including analytical sensitivity, reportable range, precision, accuracy and specificity were assessed using experimental data and data compiled from phase 1 volunteer infection studies conducted between 2013 and 2019. Guidelines for validation of laboratory-developed molecular assays were followed. Results The reportable range was 1.50 to 6.50 log10 parasites/mL with a limit of detection of 2.045 log10 parasites/mL of whole blood based on a parasite diluted standard series over this range. The assay was highly reproducible with minimal intra-assay (SD = 0.456 quantification cycle (Cq) units [0.137 log10 parasites/mL] over 21 replicates) and inter-assay (SD = 0.604 Cq units [0.182 log10 parasites/mL] over 786 qPCR runs) variability. Through an external quality assurance program, the QIMR assay was shown to generate accurate results (quantitative bias + 0.019 log10 parasites/mL against nominal values). Specificity was 100% after assessing 164 parasite-free human blood samples. Conclusions The 18S rRNA gene qPCR assay is specific and highly reproducible and can provide reliable and accurate parasite quantification. The assay is considered fit for use in evaluating drug efficacy in malaria clinical trials.

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K-ras Point Mutation Detection in Lung Cancer: Comparison of Two Approaches to Somatic Mutation Detection Using ARMS Allele-specific Amplification

Clinical Chemistry ◽

10.1093/clinchem/46.12.1929 ◽

2000 ◽

Vol 46 (12) ◽

pp. 1929-1938 ◽

Cited By ~ 53

Author(s):

Simon J Clayton ◽

Frank M Scott ◽

Jill Walker ◽

Kay Callaghan ◽

Kemal Haque ◽

...

Keyword(s):

Point Mutation ◽

Mutation Detection ◽

Lung Tumor ◽

Molecular Techniques ◽

Clinical Samples ◽

Analytical Sensitivity ◽

Real Time Quantitative Pcr ◽

Allele Specific ◽

Specific Amplification ◽

Tumor Dna

Abstract Background: The use of sensitive molecular techniques to detect rare cells in a population is of increasing interest to the molecular pathologist, but detection limits often are poorly defined in any given molecular assay. We combined the approaches of real-time quantitative PCR with ARMSTM allele-specific amplification in a novel assay for detecting mutant K-ras sequences in clinical samples. Methods: ARMS reactions were used to detect seven commonly occurring mutations in the K-ras oncogene. These mutations produce amino acid changes in codon 12 (Gly to Ala, Arg, Asp, Cys, Ser, or Val) and codon 13 (Gly to Asp). A control reaction was used to measure the total amount of amplifiable K-ras sequence in a sample so that the ratio of mutant to wild-type sequence could be measured. Quantitative data were confirmed for a selection of samples by an independent cloning and sequencing method. The assay was used to analyze 82 lung tumor DNA samples. Results: The assay detected K-ras mutations in 44% of adenocarcinomas, which is equivalent to frequencies reported in the literature using ultrasensitive techniques. Forty-six percent of squamous carcinomas were also positive. The ratio of mutant sequence in the tumor DNA samples was 0.04–100%. Conclusions: The assay is homogeneous, with addition of tumor DNA sample being the only step before results are generated. The quantitative nature of the assay can potentially be used to define the analytical sensitivity necessary for any specified diagnostic application of K-ras (or other) point mutation detection.

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Estimating Giardia cyst viability using RT-PCR

Water Science & Technology Water Supply ◽

10.2166/ws.2002.0092 ◽

2002 ◽

Vol 2 (3) ◽

pp. 107-115 ◽

Cited By ~ 1

Author(s):

M. Maux ◽

I. Bertrand ◽

C. Gantzer ◽

J. Schwartzbrod

Keyword(s):

Nested Pcr ◽

Target Genes ◽

Molecular Techniques ◽

Giardia Cyst ◽

Health Issues ◽

Structural Protein ◽

Gene Encoding ◽

Response To Stress ◽

Pcr Method ◽

Giardia Cysts

The aim of this work was to evaluate the use of molecular techniques for the detection of viable Giardia cysts in the environment to assess public health issues. Three target genes were selected: the heat shock protein gene, HSP70, which is expressed in response to stress; the giardin gene, which encodes a structural protein; and, alcohol dehydrogenase E (ADHE), a novel gene encoding an enzyme involved in the metabolism of energy. We tested the efficiency of five protocols for the extraction of either genomic DNA or total RNA from Giardia cysts: two of these protocols were previously cited in the literature and three consisted of commercial DNA extraction kits. The brands of enzyme were determined according to the primers chosen and the amplification conditions were optimised: 2.5 mM MgCl2, 0.5 mM primers and 60°C for annealing temperature. A semi-nested PCR method and an RT semi-nested PCR procedure were developed to detect mRNA from these three genes and to estimate the viability of Giardia cysts.

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Application of Nested Polymerase Chain Reaction to Detection of Salmonella in Poultry Environment

Journal of Food Protection ◽

10.4315/0362-028x-65.8.1227 ◽

2002 ◽

Vol 65 (8) ◽

pp. 1227-1232 ◽

Cited By ~ 36

Author(s):

TONGRUI LIU ◽

KAREN LILJEBJELKE ◽

ELIZABETH BARTLETT ◽

CHARLES HOFACRE ◽

SUSAN SANCHEZ ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Nested Pcr ◽

Virulence Gene ◽

Pcr Primers ◽

Processing Plant ◽

Chain Reaction ◽

Chi Square ◽

Nested Polymerase Chain Reaction ◽

Secondary Enrichment ◽

Polymerase Chain

Isolation of Salmonella from environmental and processing-plant poultry samples requires the sampling of large numbers of areas within the poultry house or plant. Subsequently, the required number of samples necessitates a large volume of work for a microbiology laboratory, especially when the protocol requires the inclusion of a delayed secondary enrichment for the isolation of Salmonella. This study examined the use of the polymerase chain reaction (PCR) to identify those secondary enrichments containing Salmonella. The unique Salmonella virulence gene invA was chosen as the target for the development of a nested PCR because of its uniform distribution among Salmonella serotypes. The use of nested PCR primers increased the sensitivity of detection 100-fold, resulting in the detection of as few as four cells. There was a strong, statistically significant positive correlation between PCR and culture results as determined by chi-square (P < 0.001) and kappa (k = 0.915; excellent agreement) tests. Using PCR to screen primary enrichments for presumptive Salmonella contamination, we improved our efficiency at isolating Salmonella upon secondary enrichment by 20%, and no false negatives were observed. This method will not only validate the use of secondary enrichment procedures but also reduce costs and manpower required for the surveillance of Salmonella.

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Fast and Sensitive Detection of Enteropathogenic Yersinia by Immunoassays

Journal of Clinical Microbiology ◽

10.1128/jcm.02137-14 ◽

2014 ◽

Vol 53 (1) ◽

pp. 146-159 ◽

Cited By ~ 8

Author(s):

Jérôme Laporte ◽

Cyril Savin ◽

Patricia Lamourette ◽

Karine Devilliers ◽

Hervé Volland ◽

...

Keyword(s):

Yersinia Pseudotuberculosis ◽

Specific Growth ◽

Limit Of Detection ◽

Economic Cost ◽

Human Feces ◽

Content Type ◽

Highly Sensitive ◽

Pathogenic Serotypes ◽

Stool Samples ◽

Enteropathogenic Yersinia

Yersinia enterocoliticaandYersinia pseudotuberculosis, the twoYersiniaspecies that are enteropathogenic for humans, are distributed worldwide and frequently cause diarrhea in inhabitants of temperate and cold countries.Y. enterocoliticais a major cause of foodborne disease resulting from consumption of contaminated pork meat and is further associated with substantial economic cost. However, investigation of enteropathogenicYersiniaspecies is infrequently performed routinely in clinical laboratories because of their specific growth characteristics, which make difficult their isolation from stool samples. Moreover, current isolation procedures are time-consuming and expensive, thus leading to underestimates of the incidence of enteric yersiniosis, inappropriate prescriptions of antibiotic treatments, and unnecessary appendectomies. The main objective of the study was to develop fast, sensitive, specific, and easy-to-use immunoassays, useful for both human and veterinary diagnosis. Monoclonal antibodies (MAbs) directed againstY. enterocoliticabioserotypes 2/O:9 and 4/O:3 andY. pseudotuberculosisserotypes I and III were produced. Pairs of MAbs were selected by testing their specificity and affinity for enteropathogenicYersiniaand other commonly found enterobacteria. Pairs of MAbs were selected to develop highly sensitive enzyme immunoassays (EIAs) and lateral flow immunoassays (LFIs or dipsticks) convenient for the purpose of rapid diagnosis. The limit of detection of the EIAs ranged from 3.2 × 103CFU/ml to 8.8 × 104CFU/ml for pathogenic serotypes I and III ofY. pseudotuberculosisand pathogenic bioserotypes 2/O:9 and 4/O:3 ofY. enterocoliticaand for the LFIs ranged from 105CFU/ml to 106CFU/ml. A similar limit of detection was observed for artificially contaminated human feces.

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Occurrence, distribution and virulence factors of clinically important Acinetobacter species recovered from selected freshwater resources in the Eastern Cape Province, South Africa

10.21203/rs.2.14951/v1 ◽

2019 ◽

Author(s):

Mary Ayobami Adewoyin ◽

Anthony Ifeanyi Okoh

Keyword(s):

South Africa ◽

Virulence Genes ◽

Virulence Gene ◽

Molecular Techniques ◽

Reca Gene ◽

Eastern Cape Province ◽

Eastern Cape ◽

Freshwater Resources ◽

Important Species ◽

Acinetobacter Species

Abstract Background : Several Acinetobacter species live in different ecosystems such as soil, freshwater, wastewater, and solid wastes. In this study, we assessed the occurrence of A. baumannii and A. nosocomialis , which are the major two clinically important species of the genus Acinetobacter , in three freshwater resources (Great Fish, Keiskemma, and Tyhume rivers) in the Eastern Cape Province, South Africa over a one year sampling regime (April 2017 - March 2018). Presumptive Acinetobacter species were subjected to molecular identification by using Acinetobacter genus-specific primers targeting the recA gene. The confirmed Acinetobacter species were further delineated into A. baumannii and A. nosocomialis using species-specific primer sets. Similarly, virulence genes, namely; afa/draBC, epsA, fimH, OmpA, PAI, sfa/focDE , and traT in the two Acinetobacter species were also determined using molecular techniques. Result : A total of 1107 presumptive Acinetobacter isolates were recovered from the freshwater resources of which 844 was confirmed positive for the Acinetobacter genus. Of the 844 Acinetobacter isolates, 285 (77%), 219 (70.9%) and 340 (79%) were recovered from Great Fish, Keiskemma and Tyhume rivers respectively. Our finding revealed that 410 (48.58%) and 23 (2.7%) of the isolates were confirmed to be A. baumannii and A. nosocomalis , respectively. The presence of these clinically-important Acinetobacter species in the freshwater studied suggests possible contamination of the selected rivers and also that A. baumannii and A. nosocomialis can thrive in aquatic environments. Besides, 308 (75.12%) A. baumannii and 3 (13.04%) A. nosocomialis isolates exhibited one or more virulence genes out of the seven tested, whereas 102 (24.88%) and 20 (86.95%) of the A. baumannii and A. nosocomialis isolates did not harbour any virulence gene. Additionally, OmpA was the most prevalent (p<0.05) virulence gene in A. baumannii with 69 (45.10%), 52 (50.98%) and 77 (49.68%) isolates in Great Fish, Keiskamma and Tyhume rivers respectively. Conclusion : The occurrence of these pathogens in rivers which are consumed by humans and livestock, as well as being used for irrigation system constitutes a risk to public health. Keywords: Freshwater resources, Molecular characterisation, Acinetobacter species, virulence gene s.

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Analytical Sensitivity, Reproducibility of Results, and Clinical Performance of Five PCR Assays for Detecting Chlamydia pneumoniae DNA in Peripheral Blood Mononuclear Cells

Journal of Clinical Microbiology ◽

10.1128/jcm.38.7.2622-2627.2000 ◽

2000 ◽

Vol 38 (7) ◽

pp. 2622-2627 ◽

Cited By ~ 32

Author(s):

J. B. Mahony ◽

S. Chong ◽

B. K. Coombes ◽

M. Smieja ◽

A. Petrich

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Nested Pcr ◽

Chlamydia Pneumoniae ◽

Mononuclear Cells ◽

Rrna Gene ◽

Analytical Sensitivity ◽

Infected Cells ◽

Pcr Assays ◽

The 16S Rrna Gene

Chlamydia pneumoniae has been associated with atherosclerosis and coronary artery disease (CAD), and its DNA has been detected in atheromatous lesions of the aorta, carotid, and coronary arteries by a variety of PCR assays. The objective of this study was to compare the performances of five published PCR assays in the detection of C. pneumoniae in peripheral blood mononuclear cells (PBMCs) from patients with coronary artery disease. The assays included two conventional PCRs, one targeting a cloned PstI fragment and one targeting the 16S rRNA gene; two nested PCRs, one targeting the 16S rRNA gene and one targeting ompA; and a touchdown enzyme time release (TETR) PCR, targeting the 16S rRNA gene. All PCRs had similar analytical sensitivities and detected a minimum of 0.005 inclusion-forming units (IFU) of C. pneumoniae; the ompA nested PCR and the TETR PCR were slightly more sensitive and detected 0.001 IFU. Assay reproducibility was examined by testing 10 replicates of C. pneumoniae DNA by each assay. All five assays showed excellent reproducibility at high levels of DNA, with scores of 10 out of 10 for 0.01 IFU, but exhibited decreased reproducibility for smaller numbers of C. pneumoniae IFU for all tests. Pairwise comparison of test results indicated that there was a significant difference between tests (Cochran Q = 32.0, P < 0.001), with thePstI fragment (P < 0.001) and 16S rRNA (P = 0.002) assays having lower reproducibility than the nested ompA and TETR assays. To further analyze assay sensitivity, C. pneumoniae-infected U-937 mononuclear cells were added to whole blood, and extracted mononuclear-cell DNA was tested by each assay. All five assays showed similar sensitivities, detecting 15 infected cells; three assays detected 3 infected cells, while all assays were negative at the next dilution (1.5 infected cells). A striking difference in performance of the five assays was seen, however, when PBMCs from CAD patients were tested for C. pneumoniae DNA. The ompA nested PCR detected C. pneumoniae DNA in 11 of 148 (7.4%) specimens, the 16S rRNA nested PCR detected 2 positives among the 148 specimens (1.4%) (P < 0.001), and the other 3 assays detected no positive specimens (P < 0.001, compared with theompA assay). These results indicate that analytical sensitivity alone does not predict the ability of an assay to detectC. pneumoniae in whole-blood-derived PBMCs. Before standardized assays can be used in wide-scale epidemiological studies, further characterization of these assays will be required to improve our understanding of their performance in the detection of C. pneumoniae in clinical material.

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Técnicas moleculares empleadas para la detección de babesia spp. en bovinos: revisión sistemática

Competitive Risaralda, generating research alliances for development ◽

10.22517/9789587224955.4.4 ◽

2021 ◽

Author(s):

Julia Victoria Arredondo Botero ◽

Joan Mateo Ríos Marín ◽

Juan Diego Ospino Sánchez

Keyword(s):

Nested Pcr ◽

Molecular Techniques ◽

Bovine Babesiosis ◽

Serological Tests ◽

Current Systematic Review ◽

Revisión Sistemática ◽

The World ◽

Polymerase Chain ◽

Research Criteria ◽

Babesia Spp

Bovine babesiosis is one of the most important parasitic diseases in the world, it is caused by protozoa of Babesia genus and generates anemia, anorexia, weight loss, jaundice and hemoglobinuria generating loss in production and even the death of the animal. Serological tests are relatively inexpensive and efficient to detect the parasite, but the results depend on factors inherent to the animal and the state of infection. Molecular markers are more precise tools to detect the parasite. The aim of the current systematic review was to review original studies, published between 2010 and 2018, in which molecular techniques were implemented to detect the parasite. The research was carried out using PubMed, LILACS, SciELO and Science Direct databases. 196 records were identified according to the research criteria and only 47 were analyzed. The main molecular techniques reported include Polymerase chain reaction (PCR), nested PCR (nPCR) and semi nested PCR, Real time PCR (qPCR), Reverse ...

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Targeting of putative fimbrial gene for detection of S. Typhi in typhoid fever and chronic typhoid carriers by nested PCR

The Journal of Infection in Developing Countries ◽

10.3855/jidc.2561 ◽

2013 ◽

Vol 7 (07) ◽

pp. 520-527 ◽

Cited By ~ 6

Author(s):

Chandra Bhan Pratap ◽

Gopal Kumar ◽

Saurabh Kumar Patel ◽

Ajay Kumar Verma ◽

Vijay K Shukla ◽

...

Keyword(s):

Nested Pcr ◽

Target Gene ◽

Reference Strain ◽

Diagnostic Technique ◽

Salmonella Typhi ◽

Detection Sensitivity ◽

Blood Samples ◽

Flic Gene ◽

Colony Forming Units ◽

Dna Fragment

Introduction: It is important to identify Salmonella Typhi infection quickly to treat acute fever patients and to prevent transmission by chronic typhoid carriers; therefore, a very specific and sensitive diagnostic technique is highly desirable, especially in endemic areas. The objective of this study was to develop a PCR protocol targeting the putative fimbrial staA gene of S. Typhi. This is a preferred target gene that is specifically amplified in the S. Typhi serotype compared to the commonly targeted fliC gene which may also be amplified from the non-typhoidal Salmonella Munchen serotype. Methodology: A new nested PCR primer methodology was designed to target the staA gene, which is a member of the fimbrial gene family specific to Salmonella Typhi only. Results: The primers were found to be very specific as the desired amplicon (377 bp) could be generated exclusively from S. Typhi strains including the reference strain (MTCC 3216) and 78 clinical isolates . Restriction digestion with HinfI confirmed the identity of the amplified DNA fragment in clinical specimens of S. Typhi origin. Furthermore, these primers were able to detect a minimum of three colony forming units per ml (1fg) in spiked blood samples. The detection sensitivity of the described primers is comparable to that of previously published primers targeting fliC gene sequences. Conclusions: This study indicates that the primers targeting the putative fimbrial staA gene are very specific to the Typhi serotype and may be a better alternative to fliC targeted amplification based diagnosis.

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Nested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples

PLoS ONE ◽

10.1371/journal.pone.0255914 ◽

2021 ◽

Vol 16 (8) ◽

pp. e0255914

Author(s):

Monia Ardhaoui ◽

Emna Ennaifer ◽

Anna Christina De Matos Salim ◽

Flávio Marcom Gomez ◽

Thalja Laasili ◽

...

Keyword(s):

Nested Pcr ◽

High Sensitivity ◽

Epidemiological Studies ◽

Multiple Infection ◽

Clinical Samples ◽

Analytical Sensitivity ◽

Hpv Genotyping ◽

Genotyping Method ◽

Specificity And Sensitivity ◽

Hpv Types

The most used methodologies for HPV genotyping in Tunisian studies are based on hybridization that are limited to a restricted number of HPV types and to a lack of specificity and sensitivity for same types. Recently, Next-Generation sequencing (NGS) technology has been efficiently used for HPV genotyping. In this work we designed and validated a sensitive genotyping method based on nested PCR followed by NGS. Eighty-six samples were tested for the validation of an HPV genotyping assay based on Nested-PCR followed by NGS. These include, 43 references plasmids and 43 positive HPV clinical cervical specimens previously evaluated with the conventional genotyping method: Reverse Line Hybridization (RLH). Results of genotyping using NGS were compared to those of RLH. The analytical sensitivity of the NGS assay was 1GE/μl per sample. The NGS allowed the detection of all HPV types presented in references plasmids. On the clinical samples, a total of 19 HPV types were detected versus 14 types using RLH. Besides the identification of more HPV types in multiple infection (6 types for NGS versus 4 for RLH), NGS allowed the identification of HPV types that were not detected by RLH. In addition, the NGS assay detected newly HPV types that were not described in Tunisia so far: HPV81, HPV43, HPV74, and HPV62. The high sensitivity and specificity of NGS for HPV genotyping in addition to the identification of new HPV types may justify the use of such technique to provide with high accuracy the profile of circulating types in epidemiological studies.

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