Epstein-Barr virus (EBV)DNA load values were measured in samples of whole blood (n ‫؍‬ 60) and plasma (n ‫؍‬ 59) by TaqMan PCR and in samples of peripheral blood lymphocytes (PBLs) (n ‫؍‬ 60) by competitive PCR (cPCR). The samples were obtained from 44 transplant recipients. The whole-blood and PBL loads correlated highly (r 2 > 0.900), whereas the plasma and PBL loads correlated poorly (r 2 ‫؍‬ 0.512). Testing of whole blood by TaqMan PCR is an acceptable alternative to testing of PBLs by cPCR for quantifying EBV DNA load.

A real time TaqMan PCR assay allows rapid identification of patients with primary EBV infection and those with EBV infectious mononucleosis.

A PCR-based assay for Bordetella pertussis was inhibited by using a calcium alginate fiber-tipped swab with an aluminum shaft but not by using a Dacron fiber-tipped swab with a plastic shaft. The calcium alginate fiber component inhibited the assay following storage for less than 1 min in a suspension of 103 CFU of B. pertussis per ml, whereas the aluminum shaft component required storage for at least 48 h in order to cause inhibition. We recommend the Dacron swab over the calcium alginate swab for collecting specimens for testing in PCR-based assays. Recent studies (5, 6, 9) suggest that PCR-based assays are more sensitive than culture for detection of Bordetella pertussis in nasopharyngeal specimens. Primers used for the amplification of target regions within the chromosome of B. pertussis and probes used for the detection of the amplified products (5, 6, 9, 14) have been developed from the DNA sequence information that is available for a repeated chromosomal element (5) and the pertussis toxin gene (10). The PCR-based assays have the potential for same-day turnaround time, whereas final isolation and identification with culture generally take 3 to 5 days. Since antibiotic susceptibility testing is not routinely performed with isolates of B. pertussis, the PCR may eliminate or reduce the need for culture. Nasopharyngeal swab and aspirate specimens are used with culture and PCR-based assays for detection of B. pertussis. For isolation of B. pertussis by culture, calcium alginate swabs are superior to Dacron, rayon, and cotton swabs (4, 7, 8). However, the effect of different swab types on PCR-based detection of B. pertussis is unknown. Suspensions of bordetellae were used to assess the effects of calcium alginate and Dacron swabs on a PCR-based assay for B. pertussis. Calcium alginateand Dacron fiber-tipped nasopharyngeal swabs and swab components were obtained from Medical Packaging Corp. (Camarillo, Calif.). The calcium alginate fiber was attached to an aluminum shaft, and the Dacron fiber was attached to a plastic (polystyrene) shaft. A recent clinical isolate of B. pertussis (strain BP5) was cultivated for 72 h on charcoal agar (Oxoid Unipath Ltd., Basingstoke, Hampshire, England) containing 10% defibrinated sheep blood. The bordetellae were harvested, washed three times by centrifugation at 2,000 x g, and suspended in sterile saline to provide suspensions with desired concentrations of the bordetellae. Template DNA was obtained from suspensions of bordetellae by digestion with lysozyme and proteinase K, and the chromosomal DNA was purified by extraction with phenol

One-step , real-time PCR assays for rhinovirus have been developed for a limited number of PCR amplification platforms and chemistries , and some exhibit cross-reactivity with genetically similar enteroviruses. We developed a one-step , real-time PCR assay for rhinovirus by using a sequence detection system (Applied Biosystems; Foster City , CA). The primers were designed to amplify a 120-base target in the noncoding region of picornavirus RNA , and a TaqMan (Applied Biosystems) degenerate probe was designed for the specific detection of rhinovirus amplicons. The PCR assay had no cross-reactivity with a panel of 76 nontarget nucleic acids , which included RNAs from 43 enterovirus strains. Excellent lower limits of detection relative to viral culture were observed for the PCR assay by using 38 of 40 rhinovirus reference strains representing different serotypes , which could reproducibly detect rhinovirus serotype 2 in viral transport medium containing 10 to 10,000 TCID 50 (50% tissue culture infectious dose endpoint) units/ml of the virus. However, for rhinovirus serotypes 59 and 69, the PCR assay was less sensitive than culture. Testing of 48 clinical specimens from children with cold-like illnesses for rhinovirus by the PCR and culture assays yielded detection rates of 16.7% and 6.3%, respectively. For a batch of 10 specimens, the entire assay was completed in 4.5 hours. This real-time PCR assay enables detection of many rhinovirus serotypes with the Applied Biosystems reagent-instrument platform. Rhinoviruses are the most common cause of viral upper respiratory tract infections and have been associated with more severe lower tract infections in compromised patients.1-3 Several real-time, RT-PCR assays have been developed; these have improved the diagnosis of rhinovirus infection over traditional culture methods, which are slow and insensitive. 4 -7 However, only a few published PCR assays have combined reverse transcription and PCR in the same real-time reaction (ie, one-step assay). 8,9 The advantages of the one-step assay over the two-step assay include improved workflow, reduction in assay preparation time, and elimination of cross contamination from the transfer of cDNA from the reverse transcription reaction into the PCR reaction. One-step assays have been developed for a limited number of PCR amplification platforms and chemistries and may not necessarily perform optimally with other platforms.8 In addition, cross-reactivity with genetically similar enteroviruses has been reported with some assays. 8,9A one-step, real-time PCR assay has not been described for the ABI Prism Sequence Detection System (Applied Biosystems; Foster City, CA) though this platform is widely used in clinical and research laboratories. High PCR efficiency with this platform generally requires the use of primers and a TaqMan probe (Applied Biosystems) with melting temperatures of 58°C to 60°C and 68°C to 70°C, respectively, and an amplicon size of 50 to 150 bp. The 5Ј noncoding region of the rhinovirus genome is most commonly targe...