Comparison and Evaluation of Real-Time PCR, Real-Time Nucleic Acid Sequence-Based Amplification, Conventional PCR, and Serology for Diagnosis of Mycoplasma pneumoniae

Abstract

Mycoplasma pneumoniae is a common cause of community-acquired pneumonia and lower-respiratory-tract infections. Diagnosis has traditionally been obtained by serological diagnosis, but increasingly, molecular techniques have been applied. However, the number of studies actually comparing these assays is limited. The development of a novel duplex real-time PCR assay for detection of M. pneumoniae in the presence of an internal control real-time PCR is described. In addition, real-time nucleic acid sequence-based amplification (NASBA) on an iCycler apparatus is evaluated. These assays were compared to serology and a conventional PCR assay for 106 clinical samples from patients with lower-respiratory-tract infection. Of the 106 samples, 12 (11.3%) were positive by all the molecular methods whereas serology with acute sample and convalescent samples detected 6 (5.6%) and 9 (8.5%), respectively. Clinical symptoms of the patients with Mycoplasmapositive results were compared to those of the other patients with lower-respiratory-tract infections, and it was found that the results for mean lower age numbers as well as the presence of chills, increased erythrocyte sedimentation rate, and raised C-reactive protein levels showed significant differences. Molecular methods are superior for diagnosis of M. pneumoniae, providing more timely diagnosis. In addition, using real-time methods involves less hands-on time and affords the ability to monitor the reaction in the same tube.

Novel Technique of Quantitative Nested Real-Time PCR Assay for Mycobacterium tuberculosis DNA

“…Although many previous studies have used a conventional single-step real-time PCR assay to quantitatively detect various infectious pathogens in different clinical samples, few have been able to describe accurate copy numbers of the causative pathogens (3,4,5,6,9,15,19,21,23). In many previous studies, even though various internal controls have been used for monitoring PCR assay conditions, they have regrettably never been used for correctly determining the copy numbers of the causative pathogens (5,6,9,15,19,21,23). To use an internal control as a "ruler" for correcting the copy number of a causative pathogen, it is necessary to construct a "new" specific internal control with equivalent amplification and detection efficiencies against the causative pathogen.…”

“…Recently, real-time PCR assays have been applied in routine diagnostic laboratory testing (1,10,14,16,22). In addition to conventional qualitative analysis, real-time PCR assays make it possible to perform accurate quantitative analyses with a high degree of reproducibility (3,4,5,6,9,15,19,21,23).…”

Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma pneumoniae

“…More rapid, higher sensitivity methods were therefore developed, one of them being the PCR for fragments of the P1 gene or the 16S rRNA gene (Dorigo-Zetsma et al, 1999;Tjhie et al, 1994;Ieven et al, 1996). In the last few years, real-time PCR methods for the diagnosis of M. pneumoniae have been described (Hardegger et al, 2000;Ursi et al, 2003;Templeton et al, 2003). The advantages of this application, compared to conventional PCR methods, are higher speed and less handling of PCR products such as electrophoretic analysis.…”

Comparison and evaluation of diagnostic techniques of mycobacterium tuberculosis in Iraq

“…These results were in accordance with those results reported by Raviglion, 21 who proposed that the real-time PCR assay with internal control achieved a sensitivity of 96.2 % and specificity of 99.2%. Templeton et al, 22 explains that internal control reaction has been included to determine the robustness of the PCR resulting by monitoring the nucleic acid extraction as well as the presence of inhibitors.…”