Dataset Information

Detection of Mycoplasma genitalium by PCR amplification of the 16S rRNA gene.

ABSTRACT: In order to develop a species-specific PCR for the detection of Mycoplasma genitalium, the sequence of 1,490 bases of the 16S rRNA gene was determined for M. genitalium G37 (type strain) and four Danish isolates of M. genitalium. The sequences of the four Danish strains, mutually different with respect to their MgPa gene, were 100% homologous, although they carried a single common base substitution compared to the type strain. Among members of the Mycoplasma pneumoniae phylogenetic cluster, M. genitalium showed the most-prominent homology to the 16S rRNA sequence of M. pneumoniae (98% homology). From regions showing the least homology to the M. pneumoniae 16S rRNA gene sequence, primers were chosen to amplify DNA from M. genitalium only. Two sets of primers were selected for their ability to detect <10 to 50 M. genitalium genome copies without cross-reactions with M. pneumoniae. The performance of these primers was compared to the performance of two pairs of primers amplifying parts of the MgPa adhesin gene; 1,030 randomly selected specimens submitted for Chlamydia trachomatis culture were screened with one of the 16S rRNA gene primer sets. A total of 41 specimens were found to be positive for this gene; 40 of these could be confirmed by one of the MgPa primer sets, whereas the other MgPa primer set detected only 21 positive specimens out of 40. These results indicate that estimates of the prevalence of M. genitalium in various populations using MgPa PCR primers could be incorrectly low if the PCR primers are located in variable regions of the MgPa gene.

SUBMITTER: Jensen JS

PROVIDER: S-EPMC149599 | biostudies-literature | 2003 Jan

REPOSITORIES: biostudies-literature

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Detection of Mycoplasma genitalium by PCR amplification of the 16S rRNA gene.

Jensen Jørgen Skov JS Borre Martin B MB Dohn Birthe B

Journal of clinical microbiology 20030101 1

In order to develop a species-specific PCR for the detection of Mycoplasma genitalium, the sequence of 1,490 bases of the 16S rRNA gene was determined for M. genitalium G37 (type strain) and four Danish isolates of M. genitalium. The sequences of the four Danish strains, mutually different with respect to their MgPa gene, were 100% homologous, although they carried a single common base substitution compared to the type strain. Among members of the Mycoplasma pneumoniae phylogenetic cluster, M. g ...[more]

PMID: 12517858

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Project description:BackgroundDiagnosis of infected individuals with Mycoplasma genitalium (MG) is often performed by real-time PCR or transcription-mediated amplification (TMA). A limitation of the MG-TMA assay is the relatively short time span of 24 h in which the collected urine is required to be transferred into a Urine Specimen Transport Tube, according to the manufacturer's guidelines. If not transferred within 24 h, the manufacturer's claimed sensitivity cannot be guaranteed anymore, and samples may instead be tested with an in-house validated real-time PCR, despite its recognized lower sensitivity. This study aimed to validate an exception to the sample transport and storage conditions of the MG-TMA assay as set by the manufacturer, being the prolongation of the acceptable testing time limit of 24 h.MethodsFrom June to December 2022, first-void urines were collected from clients attending the clinic for sexual health in Amsterdam, the Netherlands. Urine samples that tested positive for MG by TMA assay at the day of collection were concomitantly stored at room (18-24 °C) and refrigerator temperature (4-8 °C) for 15 days. The stored urine samples were tested with both an in-house validated real-time PCR and MG-TMA assay after transfer of the original urine samples to the respective test tubes at 3, 7, 12 and 15 days post collection.ResultsIn total, 47 MG-positive urine samples were collected, stored and tested for MG by real-time PCR and TMA assays. After storage at room temperature, the MG-detection rate by TMA was significantly higher compared to real-time PCR, at days 0 (p ≤ 0.001), 7 (p ≤ 0.001) and 12 (p < 0.05). After storage at refrigerator temperature, the MG-detection rate determined by TMA assay was significantly enhanced in comparison with real-time PCR at days 3 (p < 0.01), 7 (p ≤ 0.001) and 15 (p < 0.01).ConclusionsThis validation study showed that the MG-TMA assay has a superior detection rate in urine compared to real-time PCR, up to 15 days post sample collection and irrespective of storage temperature. Accepting urines older than 24 h to be tested by TMA will improve clinical diagnosis of MG infections.

Project description:BackgroundTraditional drug susceptibility testing cannot be performed in clinical laboratories due to the slow-growing characteristics of Mycoplasma genitalium when cultured in vitro. Sanger sequencing is the standard method for detecting drug resistance-associated mutations. It has been used in some laboratories to guide the choice of macrolide antibiotics for Mycoplasma genitalium infected patients. Furthermore, resistance to fluoroquinolone has become another emerging clinical challenge.ObjectiveSequencing analysis can detect unknown mutations, but it is time-consuming, requires professional analytical skills and the appropriate testing equipment. The main objective of this study was to establish a nested real-time PCR method for the simultaneous detection of 23S rRNA and parC genotypes in relation to the macrolide and fluoroquinolone resistance.Results105 MG-positive samples and 27 samples containing other pathogens were used for validation. The limit of the nested real-time PCR detection was 500 copies/reaction and there was no cross-reaction with Ureaplasma urealyticum, Mycoplasma hominis, Chlamydia trachomatis, Neisseria gonorrhoeae, Human papillomavirus, Herpes simplex virus, Candida albicans and Ureaplasma parvum, but the 23S rRNA assay cross-reacted with Mycoplasma pneumoniae. Compared with sequencing results, the sensitivity of 23S rRNA was 100% (95% CI; 93.3 -100), the specificity was 94.3% (95% CI; 79.4 - 99.0), the overall consistency was 98% (95% CI; 92.5 - 99.7) and kappa value was 0.96 (P < 0.001); the sensitivity of parC was 100% (95% CI; 93.4 - 100), the specificity was 89.7% (95% CI; 71.5 - 97.3) and the overall consistency was 96.9% (95% CI; 90.7 - 99.2) with a kappa value of 0.92 (P < 0.001).ConclusionsThe results of this sensitive and rapid alternative for identifying resistant genotypes of Mycoplasma genitalium are intuitive and easy to interpret, especially for mixed MG populations. Although the relevant 23S rRNA primers need further adjustment, this reliable method would provide an effective diagnostic tool for the selection of antibiotics in clinical practice.

Project description:The advent of next generation sequencing has coincided with a growth in interest in using these approaches to better understand the role of the structure and function of the microbial communities in human, animal, and environmental health. Yet, use of next generation sequencing to perform 16S rRNA gene sequence surveys has resulted in considerable controversy surrounding the effects of sequencing errors on downstream analyses. We analyzed 2.7×10(6) reads distributed among 90 identical mock community samples, which were collections of genomic DNA from 21 different species with known 16S rRNA gene sequences; we observed an average error rate of 0.0060. To improve this error rate, we evaluated numerous methods of identifying bad sequence reads, identifying regions within reads of poor quality, and correcting base calls and were able to reduce the overall error rate to 0.0002. Implementation of the PyroNoise algorithm provided the best combination of error rate, sequence length, and number of sequences. Perhaps more problematic than sequencing errors was the presence of chimeras generated during PCR. Because we knew the true sequences within the mock community and the chimeras they could form, we identified 8% of the raw sequence reads as chimeric. After quality filtering the raw sequences and using the Uchime chimera detection program, the overall chimera rate decreased to 1%. The chimeras that could not be detected were largely responsible for the identification of spurious operational taxonomic units (OTUs) and genus-level phylotypes. The number of spurious OTUs and phylotypes increased with sequencing effort indicating that comparison of communities should be made using an equal number of sequences. Finally, we applied our improved quality-filtering pipeline to several benchmarking studies and observed that even with our stringent data curation pipeline, biases in the data generation pipeline and batch effects were observed that could potentially confound the interpretation of microbial community data.

Project description:PCR amplification of 16S rRNA genes is a critical yet underappreciated step in the generation of sequence data to describe the taxonomic composition of microbial communities. Numerous factors in the design of PCR can impact the sequencing error rate, the abundance of chimeric sequences, and the degree to which the fragments in the product represent their abundance in the original sample (i.e., bias). We compared the performance of high fidelity polymerases and various numbers of rounds of amplification when amplifying a mock community and human stool samples. Although it was impossible to derive specific recommendations, we did observe general trends. Namely, using a polymerase with the highest possible fidelity and minimizing the number of rounds of PCR reduced the sequencing error rate, fraction of chimeric sequences, and bias. Evidence of bias at the sequence level was subtle and could not be ascribed to the fragments' fraction of bases that were guanines or cytosines. When analyzing mock community data, the amount that the community deviated from the expected composition increased with the number of rounds of PCR. This bias was inconsistent for human stool samples. Overall, the results underscore the difficulty of comparing sequence data that are generated by different PCR protocols. However, the results indicate that the variation in human stool samples is generally larger than that introduced by the choice of polymerase or number of rounds of PCR.IMPORTANCE A steep decline in sequencing costs drove an explosion in studies characterizing microbial communities from diverse environments. Although a significant amount of effort has gone into understanding the error profiles of DNA sequencers, little has been done to understand the downstream effects of the PCR amplification protocol. We quantified the effects of the choice of polymerase and number of PCR cycles on the quality of downstream data. We found that these choices can have a profound impact on the way that a microbial community is represented in the sequence data. The effects are relatively small compared to the variation in human stool samples; however, care should be taken to use polymerases with the highest possible fidelity and to minimize the number of rounds of PCR. These results also underscore that it is not possible to directly compare sequence data generated under different PCR conditions.

Dataset Information

Detection of Mycoplasma genitalium by PCR amplification of the 16S rRNA gene.

Publications

Detection of Mycoplasma genitalium by PCR amplification of the 16S rRNA gene.

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