A convenient approach to the generation of multiple internal control DNA for a panel of real-time PCR assays

Stöcher, Markus; Leb, Victoria; Berg, Jörg

doi:10.1016/s0166-0934(02)00266-5

Cited by 40 publications

(35 citation statements)

References 16 publications

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“…37,38 The IC contained a stretch of the bacterial neomycin phosphotransferase gene (neo) (GenBank Accession No. V00618, bp 452-603), which was flanked in 5Ј and 3Ј by the three forward and reverse primer binding sites specific for each PCR of the set ( Figure 1).…”

Section: Multiple Internal Control (Ic)mentioning

confidence: 99%

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

Raggam¹,

Leitner²,

Berg

et al. 2005

The Journal of Molecular Diagnostics

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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 lowpositive 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.

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Section: Multiple Internal Control (Ic)mentioning

confidence: 99%

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

Raggam¹,

Leitner²,

Berg

et al. 2005

The Journal of Molecular Diagnostics

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“…Internal controls can compensate for the differences in DNA extraction efficiency between specimens, and possible PCR inhibition in the reaction mixture [14,[29][30][31] . In addition, standardization with international reference standards is also needed for wide acceptance of this assay.…”

Section: Discussionmentioning

confidence: 99%

Comparison of real-time polymerase chain reaction with the COBAS Amplicor test for quantitation of hepatitis B virus DNA in serum samples

Shi¹,

Zhang²,

Zhu³

et al. 2008

WJG

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INTRODUCTIONIt is estimated that 350 million individuals are chronically infected with hepatitis B virus (HBV) worldwide, and about one-third of these live in China [1] . HBV infection can cause chronic hepatitis, cirrhosis and hepatocellular carcinoma [2] . Among patients with active viral replication, cirrhosis will develop in 15%-20% within 5 year [3] , and 70-90 percent of cases of hepatocellular carcinoma occur against a background of cirrhosis [4] . Quantitation of HBV DNA in serum or plasma has been an important tool in identifying individuals with active hepatitis B, to monitor the efficiency of antiviral treatment, and to predict whether the treatment will be successful [5][6][7][8][9] .Several commercial molecular assays have been developed for quantitation of HBV DNA in serum or plasma samples. One of these assays is the COBAS Amplicor HBV Monitor, which is based on the amplification of DNA targets by the use of HBV-specific primers. Other methods, such as the VERSANT HBV DNA 3.0 assay, which is based on branched-DNA signal amplification, and Hybrid Capture Ⅱ, which is based on hybridization of a chemiluminescent probe, are also routinely used in diagnostic laboratories [10,11] . However, the costs of these assays are too high to be used in developing countries such as China, and in these countries the HBV burden is much heavier than in developed countries.Recently, quantitative real-time PCR has been used to detect and quantify HBV levels in serum or plasma samples. This assay is based on the relationship between the initial DNA target levels and the threshold cycle (Ct) of the amplification products. The Ct value is smaller when the initial DNA target level is higher. Several evaluation studies have shown that real-time PCR has a higher sensitivity, a broader dynamic range, and an accurate quantitation of HBV DNA compared to those of the existing commercial methods [12][13][14][15] .The Fosun real-time PCR HBV kit is a commercial METHODS:The reference sera of the Chinese National Institute for the Control of Pharmaceutical and Biological Products and the National Center for Clinical Laboratories of China, and 158 clinical serum samples were used in this study. The linearity, accuracy, reproducibility, assay time, and costs of the real-time PCR were evaluated and compared with those of the Cobas Amplicor test. RESULTS:The intra-assay and inter-assay variations of the real-time PCR ranged from 0.3% to 3.8% and 1.4% to 8.1%, respectively. The HBV DNA levels measured by the real-time PCR correlated very well with those obtained with the COBAS Amplicor test (r = 0.948). The real-time PCR HBV DNA kit was much cheaper and had a wider dynamic range. CONCLUSION:The real-time PCR assay is an excellent tool for monitoring of HBV DNA levels in patients with chronic hepatitis B.

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“…As IAC DNA we used the previously described multiple IAC that was generated for a panel of virus-specific real-time PCR assays with competitive IACs (10 ). We show that the obtained phage IAC contains one IAC DNA fragment, is resistant to DNase I digestion, and exhibits improved storage and handling properties as well as reliable amplification in the respective competitive real-time PCR assays.…”

mentioning

confidence: 99%

“…1A); PCR conditions were exactly as described by Stö cher et al (10 ). In Clinical Chemistry 50, No.…”

mentioning

confidence: 99%

“…Equal amounts of purified phage IAC DNA and plasmid IAC DNA were examined in parallel. After DNase I treatment, sample volumes were brought to 200 L, and samples were subjected to automated DNA purification and analyzed by real-time PCR with the outermost primers for the IAC DNA [for PCR conditions and primer sequences, see Stö cher et al (10 )]. As shown in Fig.…”

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Internal Control DNA for PCR Assays Introduced into Lambda Phage Particles Exhibits Nuclease Resistance

Stöcher

Berg

2004

Clinical Chemistry

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A convenient approach to the generation of multiple internal control DNA for a panel of real-time PCR assays

Cited by 40 publications

References 16 publications

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

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

Comparison of real-time polymerase chain reaction with the COBAS Amplicor test for quantitation of hepatitis B virus DNA in serum samples

Internal Control DNA for PCR Assays Introduced into Lambda Phage Particles Exhibits Nuclease Resistance

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