Quantification of <i>Bordetella pertussis</i> in clinical samples by colorimetric detection of competitive PCR products

Erlandsson, Ann; Bäckman, Anders; Nygren, Malin; Lundeberg, Joakim; Olcén, Per

doi:10.1111/j.1699-0463.1998.tb00256.x

Cited by 8 publications

(6 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The precision of our PCR-ELISA can be considered good, as the intra-assay variation was lower than 10% and the interassay variation was around 10 to 20%, percentages which are similar to, or even lower than, those for other PCR assays based on similar principles (12,29).…”

Section: Discussionsupporting

confidence: 52%

Development and Evaluation of a PCR-Enzyme-Linked Immunosorbent Assay for Diagnosis of Human Brucellosis

Morata¹,

Queipo-Ortuño²,

Reguera³

et al. 2003

J Clin Microbiol

View full text Add to dashboard Cite

In order to overcome some of the limitations of conventional microbiological techniques in the diagnosis of human brucellosis, a simple PCR-enzyme-linked immunosorbent assay (PCR-ELISA) was developed. After amplification of a 223-bp sequence of a gene that codes for the synthesis of an immunogenetic membrane protein specific for the Brucella genus (BCSP31), the digoxigenin-labeled amplified product was hybridized with a biotinylated capture probe which was complementary to the inner part of the amplicon. The hybrid was captured on streptavidin-coated microtiter plates and detected by using an antidigoxigenin Fab-peroxidase conjugate. The detection limit of the PCR-ELISA in a background of 3.5 g of human genomic DNA was 10 fg (two bacterial cells). The PCR-ELISA showed an analytical sensitivity higher than that of ethidium bromide staining and equal to that obtained by conventional PCR followed by dot blot hybridization. In 59 peripheral blood samples from 57 consecutive patients with active brucellosis and 113 control samples, the PCR-ELISA was found to be 94.9% sensitive and 96.5% specific, whereas the sensitivity of the blood culture was only 70.1%. Since the assay can be performed in 1 day, is very reproducible, is easily standardized, and avoids the risk of infection in laboratory workers, this PCR-ELISA seems to be a practical and reliable tool for the diagnosis of human brucellosis.

show abstract

Section: Discussionsupporting

confidence: 52%

Development and Evaluation of a PCR-Enzyme-Linked Immunosorbent Assay for Diagnosis of Human Brucellosis

Morata¹,

Queipo-Ortuño²,

Reguera³

et al. 2003

J Clin Microbiol

View full text Add to dashboard Cite

show abstract

“…For assessment of the sensitivity of culture, various clinical case definitions are used, and sensitivities have been calculated for various patient and age groups, such as in outbreak settings (schools), vaccine efficacy trials (infants), or a population with sporadic cases (all age groups). The sensitivity of culture also depends on the bacterial load, as demonstrated by studies using semiquantitative PCRs (40,111,(262)(263)(264). It was found that the culture sensitivity decreased with increasing C q values (and hence a decreasing bacterial DNA presence) for real-time PCR (85,265).…”

Section: Factors That Influence the Sensitivity Of Diagnostic Methodsmentioning

confidence: 99%

Laboratory Diagnosis of Pertussis

2015

View full text Add to dashboard Cite

SUMMARYThe introduction of vaccination in the 1950s significantly reduced the morbidity and mortality of pertussis. However, since the 1990s, a resurgence of pertussis has been observed in vaccinated populations, and a number of causes have been proposed for this phenomenon, including improved diagnostics, increased awareness, waning immunity, and pathogen adaptation. The resurgence of pertussis highlights the importance of standardized, sensitive, and specific laboratory diagnoses, the lack of which is responsible for the large differences in pertussis notifications between countries. Accurate laboratory diagnosis is also important for distinguishing between the several etiologic agents of pertussis-like diseases, which involve both viruses and bacteria. If pertussis is diagnosed in a timely manner, antibiotic treatment of the patient can mitigate the symptoms and prevent transmission. During an outbreak, timely diagnosis of pertussis allows prophylactic treatment of infants too young to be (fully) vaccinated, for whom pertussis is a severe, sometimes fatal disease. Finally, reliable diagnosis of pertussis is required to reveal trends in the (age-specific) disease incidence, which may point to changes in vaccine efficacy, waning immunity, and the emergence of vaccine-adapted strains. Here we review current approaches to the diagnosis of pertussis and discuss their limitations and strengths. In particular, we emphasize that the optimal diagnostic procedure depends on the stage of the disease, the age of the patient, and the vaccination status of the patient.

show abstract

“…Various PCR protocols have been developed that target different regions of the genome, e.g., insertion sequences IS481 and IS1001 (3,4,7,16,19), the pertussis toxin promoter region (7,8,19,25), the adenylate cyclase gene (2), and the porin gene (5,14). In a comparison of different PCR assays used in seven pertussis vaccine studies all assays increased sensitivity for detection of pertussis cases by at least 70% compared with culture, and false-positive results were less than 1% of the total (26).…”

mentioning

confidence: 99%

Real-Time LightCycler PCR for Detection and Discrimination of Bordetella pertussis and Bordetella parapertussis

et al. 2002

View full text Add to dashboard Cite

Real-time PCR assays based on the LightCycler technology were developed for individual (simplex PCR) and simultaneous (duplex PCR) detection and discrimination of Bordetella pertussis and Bordetella parapertussis in clinical samples. The assays were evaluated with 113 specimens from patients with and without symptoms of pertussis. Results were compared to those from conventional culture and TaqMan real-time PCR. The analytical sensitivity ranged from 0.1 to 10 CFU for B. pertussis and B. parapertussis, and intra- and interassay variations were less than 7%. Results were available within 2 h. With the simplex format, 21 of 100 samples from patients with clinical symptoms of pertussis were positive for B. pertussis and/or B. parapertussis. With the duplex format, 18 of 100 samples were positive. LightCycler PCR increased the diagnostic sensitivity over that of culture by 2.0-fold (duplex PCR) (P = 0.08) to 2.3-fold (simplex PCR) (P = 0.02). Our data suggest that duplex PCR in this format showed good analytical sensitivity but lost some sensitivity on clinical samples compared with the simplex format.

show abstract

Quantification of Bordetella pertussis in clinical samples by colorimetric detection of competitive PCR products

Cited by 8 publications

References 15 publications

Development and Evaluation of a PCR-Enzyme-Linked Immunosorbent Assay for Diagnosis of Human Brucellosis

Development and Evaluation of a PCR-Enzyme-Linked Immunosorbent Assay for Diagnosis of Human Brucellosis

Laboratory Diagnosis of Pertussis

Real-Time LightCycler PCR for Detection and Discrimination of Bordetella pertussis and Bordetella parapertussis

Contact Info

Product

Resources

About