Development of a multiplex real-time PCR assay for detection of Mycoplasma pneumoniae, Chlamydia pneumoniae and mutations associated with macrolide resistance in Mycoplasma pneumoniae from respiratory clinical specimens

Nummi, Maaret; Mannonen, Laura; Puolakkainen, Mirja

doi:10.1186/s40064-015-1457-x

Cited by 25 publications

(30 citation statements)

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“…The nucleic acid amplification techniques (NAATs) targeted towards DNA or RNA have increasingly been explored for identification of pathogens including M. pneumoniae in infectious respiratory diseases [10][11][12][13][14][15][16][17]. So far, only a few studies have described the application of NAATs for pathogen detection in children with CAP [25][26][27].…”

Section: Discussionmentioning

confidence: 99%

“…Thus, in the recent study, the M. pneumoniae was added into our previously used pathogen panel that included 20 types/subtypes of viruses [9]. Meanwhile, several other groups have also designed the multiplex-PCR for the early detection of M. pneumoniae infection [10][11][12][13][14][15][16][17], but so far, the direct comparison between multiplex-PCR and serology test in larger pediatric clinical samples has rarely been reported [6]. Besides, the M. pneumoniae RNA detection (real-time isothermal transcription-mediated RNA amplification assay, Mp-SAT) that targeted on the specific 16SrRNA has been invented and investigated [18,19], which could eliminate the false positive cases resulted from the previous infection or the nonpathogenic carrier state [20].…”

Section: Introductionmentioning

confidence: 99%

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A comparison study between GeXP-based multiplex-PCR and serology assay for Mycoplasma pneumoniae detection in children with community acquired pneumonia

et al. 2017

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BackgroundDiagnosis of community-acquired pneumonia (CAP) caused by Mycoplasma pneumoniae (Mp) in children has been hampered by difficulty in obtaining convalescent serum and time constraints. In this study, the two diagnostic assays that targeted respectively on Mp-antibody and Mp-DNA were retrospectively investigated.MethodsA total of 3146 children were clinically diagnosed to have CAP and were confirmed by chest X-ray during March 2015 to February 2016 in Children’s hospital of Hebei Province (China). Both of the sera and sputum samples were collected in 24 h after their admission. The Mp-antibody was examined by the passive particle agglutination assay and a fourfold or greater increase of antibody titers of paired sera or≧1:160 titer of single serum was set as the serology positive. Mp-DNA in the sputum samples was tested by a multiplex-PCR method named GeXP assay (multiplex PCR combined with automated capillary electrophoresis). In order to eliminate the false positive results caused by the asymptomatic carriage after infected by M. pneumoniae, the inconsistent samples were tested by the real-time isothermal transcription-mediated RNA amplification assay (SAT).ResultsThe inter-rated agreement test was performed in 3146 CAP patients, with a highest kappa value in the school-age children as 0.783. There were 6.29% (198/3146) cases showed inconsistent results determined by GeXP and serology assay. All of the 19 GeXP(+)/Serology (−) samples and a randomly chosen 27 from 179 GeXP(−)/Serology (+) samples were tested by SAT assay, and a 97.8% diagnosis agreement was observed between SAT and GeXP assay, but not with the serology assay. In addition, patients who were detected only by serology or only by multiplex-PCR were significantly younger than those with both methods positive (3.0 and 1.5 years vs. 5.0 years, p < 0.01). The Viral-Mp coinfection accounted for 37.0% (97/262), which was more common in winter and spring (p < 0.05) and in the infantile group (p < 0.01), compared to the pure Mp positive ones.ConclusionIn some children CAP cases, the Mp laboratory diagnosis was inconsistent between serology and multiplex-PCR assay. Verified by the SAT assay, the GeXP showed a more sensitive and reliable performance compared with the serology assay. Furthermore, employing the multiplex-PCR could provide more information on the associated pathogens for clinical assessment of CAP.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-017-2614-3) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A comparison study between GeXP-based multiplex-PCR and serology assay for Mycoplasma pneumoniae detection in children with community acquired pneumonia

et al. 2017

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“…When testing patient surveillance samples, primers and probe that target Homo sapiens β-globin chain (GenBank accession no. AY260740.1) 5′-GGTT GGGA TAAG GCTG GATTATT-3′, 5′-CAGGAGCTGTGGGAG-GAAGA-3′ and 5′-JOE/ZEN-CAAG CTAG GCCC TTTT GCTA ATCA TGTTCA-Iowa Black FQ-3′ were used as the internal control [8].…”

Section: Auris Real-time Pcr Assaymentioning

confidence: 99%

Optimizing a real-time PCR assay for rapid detection of Candida auris in nasal and axillary/groin samples

Malczynski

Dowllow

Rezaeian

et al. 2020

Journal of Medical Microbiology

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Introduction. Candida auris is an emerging fungal pathogen. The organism can cause invasive infections associated with high mortality, has been implicated in outbreaks in healthcare settings and is frequently resistant to multiple antifungal agents, making it a significant challenge to infection prevention and patient treatment. Aim. To implement a real-time PCR assay for detection of C. auris in patient surveillance samples collected with the Copan Liquid Amies elution swab (ESwab) collection and transport system. Methodology. We optimized a real-time PCR testing procedure based on the sample collection device used in our institution. Results . ESwab transport medium was strongly inhibitory to the real-time PCR. Removing the medium with centrifugation, followed by suspending the pellet in PBS-BSA buffer (concentration 1 %), sufficiently eliminated the inhibition. The manual sample preparation method, freeze–thaw followed by mechanical disruption, allowed the detection of C. auris at the lowest cell concentration. Conclusion . The optimized procedure was used to test 1414 patient surveillance samples. The real-time PCR detected all culture-positive samples with 100 % sensitivity and 100 % specificity.

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“…The microarray CDV detection limit was 10 viral copies, which was even lower than the 23.2 copies using CDV hemi-nested RT-PCR (Di Francesco et al, 2012). Although real time RT-PCR can achieve similar low detection limit for these respiratory pathogens (Aeschbacher et al, 2015;Decaro et al, 2010;Nummi et al, 2015;Tatti et al, 2011;Wilkes et al, 2014), the machine cost is high. The microarray presented in this study compared to real time RT-PCR could be a feasibly alternative and especially preferable to the local under-equipped laboratories.…”

Section: Discussionmentioning

confidence: 89%

The detection and differentiation of canine respiratory pathogens using oligonucleotide microarrays

Wang

Kuo

Chueh

et al. 2017

Journal of Virological Methods

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a b s t r a c tCanine respiratory diseases are commonly seen in dogs along with co-infections with multiple respiratory pathogens, including viruses and bacteria. Virus infections in even vaccinated dogs were also reported. The clinical signs caused by different respiratory etiological agents are similar, which makes differential diagnosis imperative. An oligonucleotide microarray system was developed in this study. The wild type and vaccine strains of canine distemper virus (CDV), influenza virus, canine herpesvirus (CHV), Bordetella bronchiseptica and Mycoplasma cynos were detected and differentiated simultaneously on a microarray chip. The detection limit is 10, 10, 100, 50 and 50 copy numbers for CDV, influenza virus, CHV, B. bronchiseptica and M. cynos, respectively. The clinical test results of nasal swab samples showed that the microarray had remarkably better efficacy than the multiplex PCR-agarose gel method. The positive detection rate of microarray and agarose gel was 59.0% (n = 33) and 41.1% (n = 23) among the 56 samples, respectively. CDV vaccine strain and pathogen co-infections were further demonstrated by the microarray but not by the multiplex PCR-agarose gel. The oligonucleotide microarray provides a highly efficient diagnosis alternative that could be applied to clinical usage, greatly assisting in disease therapy and control.

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Development of a multiplex real-time PCR assay for detection of Mycoplasma pneumoniae, Chlamydia pneumoniae and mutations associated with macrolide resistance in Mycoplasma pneumoniae from respiratory clinical specimens

Cited by 25 publications

References 25 publications

A comparison study between GeXP-based multiplex-PCR and serology assay for Mycoplasma pneumoniae detection in children with community acquired pneumonia

A comparison study between GeXP-based multiplex-PCR and serology assay for Mycoplasma pneumoniae detection in children with community acquired pneumonia

Optimizing a real-time PCR assay for rapid detection of Candida auris in nasal and axillary/groin samples

The detection and differentiation of canine respiratory pathogens using oligonucleotide microarrays

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