Rapid and sensitive diagnosis of cytomegalovirus and Pneumocystis carinii pneumonia in patients with haematological neoplasia by using capillary polymerase chain reaction

Abstract

We attempted the simultaneous detection of cytomegalovirus DNA (CMV-DNA) and Pneumocystis carinii (carinii-DNA) in sputum samples obtained from 20 patients with haematological neoplasm with pneumonia, using rapid cycle DNA amplification (capillary PCR). We used a thermal cycler for capillary PCR which featured recirculation of hot air for rapid temperature control of 10 microliters reaction samples in thin glass capillary tubes. We extracted DNA from patients' sputa using a simple method. A comparison of the results obtained using the phenol extraction-ethanol precipitation method and those obtained using our simple method was made, and demonstrated complete agreement between the two. For detection of CMV-DNA and P. carinii-DNA with capillary PCR it was not necessary to vary temperature setting based on the primers used. Therefore, capillary PCR was used for the simultaneous detection of CMV and P. carinii. After amplification, the total time required for which was 20 min, amplified products were electrophoresed on agarose gels and visualized with ethidium bromide. Product sensitivity was higher with capillary PCR than with conventional PCR. We conclude that capillary PCR amplification is a valuable tool for rapid and simple diagnosis of CMV and P. carinii pneumonias.

High Concentration of (1→3)-β-D-Glucan in BAL Fluid in Patients With Acute Eosinophilic Pneumonia

“…19,20 Detection of Pneumocystis carinii DNA Fragments in BALF by Capillary Polymerase Chain Reaction BALF samples were obtained from patients with AEP to detect the specific DNA fragments of P carinii by polymerase chain reaction (PCR), as previously described. 21 All BALF samples were pelleted by centrifugation at 1,600g for 10 min, after which the supernatant was removed. The pellets were resuspended in TE buffer containing 10 mmol/L Tris-HCl (pH 8.0), 100 mmol/L KCL, 2.5 mmol/L MgCl2, 1% Tween 20, 1% NP 40, and 80 g protein kinase K per milliliter for 60 min at 56°C and then were incubated for 10 min at 100°C.…”

Detection of Bordetella pertussis by rapid-cycle PCR and colorimetric microwell hybridization

“…Capillary tubes were originally used as the reaction vessel for the rapid cycling system since they have thin walls, a large surface area, and a low volume. Using this type of reaction vessel, Honda et al (13) developed a rapid procedure for detecting cytomegalovirus and Pneumocystis carinii in sputum specimens and Locher et al (18) developed a procedure for the detection of foot-and-mouth disease virus in cattle. However, capillary tubes are difficult to use because they are fragile and the ends need to be heat-sealed to prevent evaporation during amplification.…”

Evaluation of a Real-Time PCR Assay for the Diagnosis of Pneumocystis pneumonia

“…PCR was performed according to Honda et al [11]. For the detection of Pneumocystis-specific DNA, the primers PAZ102-E (5'-GATGGCTGTTTC-CAAGCCCA-3') and PAZ102-H (5'-GTGTACGTT-GCAAAGTACTC-3'), derived from the mitochondrial large subunit rRNA (mtLSUrRNA), and which amplified a 346 bp sequence, were used as reported previously [31].…”

“…The extracted DNA (described above in Preparation of DNA specimens) was used for amplification of the major surface glycoprotein (MSG) gene of Pneumocystis [14]. A PCR method previously described [11] was modified for the real-time assay. The commercially synthesized primers (Operon Biotechnologies, Inc. Tokyo, Japan) JKK14/15 (5'-GAA TGC AAA TCY TTA CAG ACA ACA G-3') and JKK17 (5'-AAA TCA TGA ACG AAA TAA CCA TTG C-3') amplified a 250-bp segment of the multicopy MSG gene family [14].…”