A method for the rapid construction of cRNA standard curves in quantitative real-time reverse transcription polymerase chain reaction

Fronhoffs, Stefan; Totzke, Gudrun; Stier, Sebastian; Wernert, Nicolas; Rothe, Marcus; Brüning, Thomas; Koch, Brigitte; Sachinidis, Agapios; Vetter, Hans; Ko, Yon

doi:10.1006/mcpr.2002.0405

Cited by 200 publications

(124 citation statements)

References 47 publications

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“…(ii) Preparation of cRNA transcript standard: Fragments containing the target regions of the assays were amplified from HIV-1 genome plasmid PNL-43, and plasmids containing the 5?-UTR sequence of HCV 1b with primers containing a T7 promoter sequence on the reverse side were transcribed in vitro using a T7 RiboMAX TM Large Scale RNA Production System (Promega, Madison, WI, USA). The synthetic cRNA transcripts were then purified and quantified using a UV absorbance method (23,24). The copy number of cRNA standards was calculated using the following formula: RNA copy number (copies/mL) ＝ RNA concentration (g/mL) × 6.02 × 10 23 (copies/mol) ÷ RNA length (nt) ÷ 345 (g/mol).…”

Section: Methodsmentioning

confidence: 99%

Development of a Multiplex Real-Time PCR Assay for the Detection of <i>Treponema pallidum</i>, HCV, HIV-1, and HBV

et al. 2015

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SUMMARY: Treponema pallidum, hepatitis C virus (HCV), human immunodeficiency virus (HIV)-1, and hepatitis B virus (HBV) are major causes of sexually transmitted diseases passed through blood contact. The development of a sensitive and efficient method for detection is critical for early diagnosis and for large-scale screening of blood specimens in China. This study aims to establish an assay to detect these pathogens in clinical serum specimens. We established a TaqMan-locked nucleic acid (LNA) realtime polymerase chain reaction (PCR) assay for rapid, sensitive, specific, quantitative, and simultaneous detection and identification. The copy numbers of standards of these 4 pathogens were quantified. Standard curves were generated by determining the mean cycle threshold values versus 10-fold serial dilutions of standards over a range of 10 6 to 10 1 copies/mL, with the lowest detection limit of the assay being 10 1 copies/mL. The assay was applied to 328 clinical specimens and compared with enzyme-linked immunosorbent assay (ELISA) and commercial nucleic acid testing (NAT) methods. The assay identified 39 T. pallidum-, 96 HCV-, 13 HIV-1-, 123 HBV-, 5 HBV/HCV-, 1 T. pallidum/HBV-, 1 HIV-1/HCV-, and 1 HIV-1/T. pallidum-positive specimens. The high sensitivity of the assay confers strong potential for its use as a highly reliable, cost-effective, and useful molecular diagnostic tool for largescale screening of clinical specimens. This assay will assist in the study of the pathogenesis and epidemiology of sexually transmitted blood diseases.

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Section: Methodsmentioning

confidence: 99%

Development of a Multiplex Real-Time PCR Assay for the Detection of <i>Treponema pallidum</i>, HCV, HIV-1, and HBV

et al. 2015

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“…The RNA transcript obtained was quantified spectrophotometrically at 260 nm (Qubit; Invitrogen). RNA copy numbers were calculated as described previously (Fronhoffs et al, 2002). Tenfold RNA transcript dilutions, ranging from 6 to 6610 7 molecules, were used to obtain standard curves.…”

Section: Methodsmentioning

confidence: 99%

Persistence of highly pathogenic avian influenza virus (H7N1) in infected chickens: feather as a suitable sample for diagnosis

Busquets

Abad

Alba

et al. 2010

Journal of General Virology

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“…The PCR products (884 bp) were purified with the Strataprep PCR purification kit (Stratagene) and DNA amounts were quantified by measuring the A 260 in a 96-well plate reader (Biotek mQuant). This amount was converted to molecule number as previously described (Fronhoffs et al, 2002). Then PCR products were 10-fold serially diluted from 5610 8 to 50 molecules ml 21 and three q-PCRs were carried out in a LightCycler apparatus (Roche) with primers 2SF and 2R, 2bSF and 2R, and 2a and 2R, generating standard curves for stx 2 , stx 2-vhb , and stx 2-vha /stx 2c respectively.…”

Section: Rna Extraction and Quantitative Real-time Pcr (Q-pcr)mentioning

confidence: 99%

Differential expression of stx2 variants in Shiga toxin-producing Escherichia coli belonging to seropathotypes A and C

Sablet¹,

Bertin²,

Vareille³

et al. 2008

Microbiology

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Only a subset of Shiga toxin (Stx)-producing Escherichia coli (STEC) are human pathogens, but the characteristics that account for differences in pathogenicity are not well understood. In this study, we investigated the distribution of the stx variants coding for Stx2 and its variants in highly virulent STEC of seropathotype A and low-pathogenic STEC of seropathotype C. We analysed and compared transcription of the corresponding genes, production of Shiga toxins, and stx-phage release in basal as well as in induced conditions. We found that the stx 2 variant was mainly associated with strains of seropathotype A, whereas most of the strains of seropathotype C possessed the stx 2-vhb variant, which was frequently associated with stx 2 , stx 2-vha or stx 2c . Levels of stx 2 and stx 2 -related mRNA were higher in strains belonging to seropathotype A and in those strains of seropathotype C that express the stx 2 variant than in the remaining strains of seropathotype C. The stx 2-vhb genes were the least expressed, in basal as well as in induced conditions, and in many cases did not seem to be carried by an inducible prophage. A clear correlation was observed between stx mRNA levels and stx-phage DNA in the culture supernatants, suggesting that most stx 2 -related genes are expressed only when they are carried by a phage. In conclusion, some relationship between stx 2 -related gene expression in vitro and the seropathotype of the STEC strains was observed. A higher expression of the stx 2 gene and a higher release of its product, in basal as well as in induced conditions, was observed in pathogenic strains of seropathotype A. A subset of strains of seropathotype C shows the same characteristics and could be a high risk to human health.

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A method for the rapid construction of cRNA standard curves in quantitative real-time reverse transcription polymerase chain reaction

Cited by 200 publications

References 47 publications

Development of a Multiplex Real-Time PCR Assay for the Detection of <i>Treponema pallidum</i>, HCV, HIV-1, and HBV

Development of a Multiplex Real-Time PCR Assay for the Detection of <i>Treponema pallidum</i>, HCV, HIV-1, and HBV

Persistence of highly pathogenic avian influenza virus (H7N1) in infected chickens: feather as a suitable sample for diagnosis

Differential expression of stx2 variants in Shiga toxin-producing Escherichia coli belonging to seropathotypes A and C

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