Gerhard Mühlbauer

Detection of Methicillin-Resistant Staphylococcus aureus and Simultaneous Confirmation by Automated Nucleic Acid Extraction and Real-Time PCR

ABSTRACT A molecular assay for the simultaneous detection of a Staphylococcus aureus-specific gene and the mecA gene, responsible for the resistance to methicillin in staphylococci, was evaluated. The assay included an automated DNA extraction protocol conducted with a MagNA Pure instrument and real-time PCR conducted with a LightCycler instrument. The performance and robustness of the assay were evaluated for a suspension of methicillin-resistant S. aureus (MRSA) strain with a turbidity equivalent to a McFarland standard of 0.5, which was found to be the ideal working concentration. The specificity of the new molecular assay was tested with a panel of 30 gram-negative and gram-positive bacterial strains other than MRSA. No cross-reactivity was observed. In a clinical study, 109 isolates of MRSA were investigated. All clinical MRSA isolates gave positive results for the S. aureus-specific genomic target, and all but one were positive for the mecA gene. In conclusion, the new molecular assay was found to be quick, robust, and laborsaving, and it proved to be suitable for a routine molecular diagnostic laboratory.

Single-Run, Parallel Detection of DNA from Three Pneumonia-Producing Bacteria by Real-Time Polymerase Chain Reaction

A molecular assay for parallel detection of three bacteria, Chlamydia (C.) pneumoniae, Legionella (L.) spp., and Mycoplasma (M.) pneumoniae, in clinical specimens by a set of real-time polymerase chain reactions (PCRs) in a single run was evaluated. Bacterial DNAs were extracted by an automated DNA extraction protocol on the MagNA Pure LC System. Amplification and detection were done by real-time PCR on the LightCycler (LC) instrument. For amplification, specific oligonucleotides derived from the 16s rRNA genes of C. pneumoniae, L. spp., and M. pneumoniae were used. The three assays were complemented with an internal control (IC), a specially designed DNA fragment which contains the specific primer binding sites for the three PCRs. The IC was added to the samples, co-extracted, and co-amplified. Primers and hybridization probes were designed to suit one LC PCR program. LC PCRs were established, detection limits were determined, and clinical samples were tested. The detection limits were found between 5.0 and 0.5 IFU/CFU per PCR reaction for each of the bacteria. A total number of 100 clinical specimens were tested for validation of the molecular assay. Tested samples included 63 bronchoalveolar lavages (BALs) and 37 induced sputa specimens. The internal control was detected in all negative and low-positive samples; no inhibition was found throughout the whole study. Additionally, samples underwent testing by culture for L. spp., and M. pneumoniae; for C. pneumoniae, the serological microimmunofluorescence (MIF) test was used. In conclusion, the developed set of LC PCR assays permits parallel detection of C. pneumoniae, L. spp., and M. pneumoniae in a single LC run. This molecular assay may lead to accurate and early diagnosis of pneumonia produced by these three types of bacteria. The assay proved to be suitable for the high-throughput routine diagnostic laboratory.

Rapid detection of enterovirus infection by automated RNA extraction and real-time fluorescence PCR

BACKGROUND Molecular detection has been shown to be superior to tissue culture for the detection of enteroviruses in cerebrospinal fluid (CSF) specimens. OBJECTIVES In this study, a qualitative molecular assay based on automated RNA extraction with the MagNA Pure LC and real-time PCR on the LightCycler (LC) instrument was evaluated and compared with an in-house molecular assay. STUDY DESIGN A total of 109 CSF specimens were investigated for the comparative study. The detection limit of the new molecular assay was determined with 10-fold dilutions of two enterovirus strains and with the Third European Union Concerted Action Enterovirus Proficiency Panel. RESULTS With the enterovirus strains, the detection limit of the LC assay was found to be 0.1 TCID(50) (50% tissue culture infective dose). When samples of the Third European Union Concerted Action Enterovirus Proficiency Panel were tested, both molecular assays gave identical results to the expected results, which were based upon the results of three reference laboratories using a total of four different molecular methods before distribution of the panel. When clinical specimens were tested, there was a correlation between the LC assay and the in-house assay in 105 of 109 cerebrospinal fluids. CONCLUSIONS The new molecular assay allows rapid detection of enterovirus RNA in CSF. It was found to be labor saving and showed sufficient sensitivity.

Single-tube two-round polymerase chain reaction using the LightCycler™ instrument

BACKGROUND For many diagnostic applications, the specificity and sensitivity of polymerase chain reaction (PCR) is markedly enhanced by applying two rounds of PCR with nested or semi-nested pairs of primers. In two-round PCR protocols on the LightCycler instrument, amplification products must be collected from the capillaries by centrifugation, a procedure thought to be particularly prone to product carry-over. OBJECTIVE Development of a technique to perform two-round PCR with the LightCycler instrument in a single closed capillary. STUDY DESIGN Silicone oil was used to separate the second-round primers from first-round PCR mixture during the first-round PCR. The feasibility of the principle was demonstrated using a semi-nested primer system for the PCR analysis of genomic DNA. The first-round PCR reaction mixture was loaded into the capillary and covered by oil. Then, the second-round PCR reaction mixture was layered on top of it. PCR was run in two rounds separated by a centrifugation step that combined the second-round PCR mixture with the first-round products. Amplified products were visualized by fluorescence melting curve analysis. RESULTS When a dilution series of genomic DNA was used for the single-capillary two-round PCR, 0.1 ng of DNA could consistently be detected. This was a 10-fold increase of sensitivity in comparison with single-round PCR. With the new technique, the first-round reaction mixture was sufficiently separated from second-round primers by the oil layer. CONCLUSIONS Two-round PCR on the LightCycler using a single closed capillary excluded the possibility of amplification product carry-over. This new technique can easily be adapted for numerous applications, and should show feasibility for many nested primer PCR applications currently in use to the clinical detection of virus-derived DNA.

Qualitative Detection of Herpes Simplex Virus DNA in the Routine Diagnostic Laboratory

Herpes simplex virus (HSV) causes a wide spectrum of clinical manifestations in the central nervous system. Neonatal HSV infection following exposure to the virus at delivery can produce severe disseminated infection and death. Effective therapeutic management exists today, however, antiviral drugs must be administered early. Therefore, rapid laboratory diagnosis is essential for decreasing the lethality as well as the sequelae of HSV infection.

Evaluation of a new assay for detection of herpes simplex virus type 1 and type 2 DNA by real-time PCR/Evaluierung eines neuen Tests zur Detektierung von Herpes simplex Virus Typ 1 und Typ 2 DNA mittels Real-time PCR

Abstract Abstract Molecular detection of herpes simplex virus (HSV) DNA is recognized as reference standard assay for the sensitive and specific diagnosis of central nervous system and genital infections caused by HSV. In this study, a qualitative molecular assay based on automated DNA extraction on the MagNA Pure LC (Roche Applied Science, Mannheim, Germany) and a commercially available kit, the LightCycler – HSV 1/2 Detection Kit (Roche), were evaluated. This kit includes a homologous internal control. The accuracy and the detection limit of the new molecular assay were determined with samples from European Union Concerted Action HSV Proficiency Panels. A total of 153 clinical specimens including cerebrospinal fluids, genital swabs, and oral swabs were investigated and the results were compared to those obtained from a home-brew molecular assay based on manual DNA extraction and real-time polymerase chain reaction (PCR). When the accuracy of the new molecular assay was tested, all positive samples except for the one containing 3.0 × 102–9.0 × 102 HSV type 1 (HSV-1) genome equivalents (GE) per ml could be detected. All negative samples tested negative. When the detection limit was determined, 6.0 × 102–1.8 × 103 HSV-1 GE per ml, i.e. 12 to 36 GE per LightCycler (LC) capillary and 2.0 × 102–6.0 × 102 HSV type 2 (HSV-2) GE per ml, i.e. 4 to 12 GE per LC capillary could consistently be detected. Results obtained from clinical specimens corresponded to 100% to those obtained by the molecular assay based on manual DNA extraction and real-time PCR. In seven clinical samples, an unexpected melting peak was detected. In conclusion, the new molecular assay allows rapid detection of HSV-1 and HSV-2 DNA in the routine diagnostic laboratory. The inclusion of a homologous internal control ensures accurate interpretation of negative results.