scholarly journals Two variants of quantitative reverse transcriptase PCR used to show differential expression of α-, β- and γ-fibrinogen genes in rat liver lobes

1997 ◽  
Vol 321 (3) ◽  
pp. 769-776 ◽  
Author(s):  
Junlong ZHANG ◽  
Mina DESAI ◽  
Susan E. OZANNE ◽  
Cora DOHERTY ◽  
C. Nicholas HALES ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Rat Liver ◽  
Coefficient Of Variation ◽  
Copy Number ◽  
Internal Standard ◽  
Rt Pcr ◽  
Total Rna ◽  
Reverse Transcriptase Pcr ◽  
Mrna Copy Number ◽  
Sensitive Method

Quantitative reverse transcriptase PCR (RT-PCR) is a sensitive method for the measurement of mRNA copy number. However, the methodology has gained a reputation for poor reproducibility, leading to concern over the validity of much of the data generated using this technique. We have developed two variants of quantitative competitive RT-PCR using a synthesized RNA as an internal standard to measure precisely the relative levels of α-, β- and γ-fibrinogen mRNAs in the four lobes of the rat liver. In the first of these variants we altered only the amount of total RNA in the RT-PCR reaction, keeping the amount of internal standard RNA and the number of PCR cycles constant. In the second variant only the number of PCR cycles was altered, and the amounts of total RNA and standard RNA were kept constant. Both variants of RT-PCR allowed calculation of the number of mRNA copies, which did not differ significantly between the two techniques. Of the two variants, the second gave better reproducibility, and the intra-assay coefficient of variation for this technique was 14% (n = 20). Using these two variants we have shown that there are different numbers of fibrinogen mRNAs in the four liver lobes for each of the three genes (α-fibrinogen F = 14.64, P = 0.0003; β-fibrinogen F = 3.74, P = 0.04; γ-fibrinogen F = 3.75, P = 0.04). In conclusion, by using two variants of quantitative competitive RT-PCR we have shown that this technique can be used to give reproducible results, and the low intra-assay coefficient of variation suggests that quantitative RT-PCR should be the technique of choice for accurate measurement of mRNA copy number.

scholarly journals First Genome Sequence of Newcastle Disease Virus of Genotype VIIi from Jordan

2018 ◽  
Vol 7 (23) ◽  
Author(s):  
Mustafa Ababneh ◽  
Helena L. Ferreira ◽  
Mohammad Khalifeh ◽  
David L. Suarez ◽  
Claudio L. Afonso
Keyword(s):  
Reverse Transcriptase ◽  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Genome Sequence ◽  
Illumina Sequencing ◽  
Newcastle Disease ◽  
Rt Pcr ◽  
Total Rna ◽  
Reverse Transcriptase Pcr ◽  
Chicken Spleen

Newcastle disease virus (NDV) was detected by reverse transcriptase PCR (RT-PCR) from total RNA isolated from a chicken spleen of a backyard flock in Jordan. The complete coding genome sequence of NDV/chicken/Jordan/J11-spleen/2018 was obtained with MiSeq (Illumina) sequencing.

scholarly journals Differential priming of RNA templates during cDNA synthesis markedly affects both accuracy and reproducibility of quantitative competitive reverse-transcriptase PCR

1999 ◽  
Vol 337 (2) ◽  
pp. 231-241 ◽  
Author(s):  
Junlong ZHANG ◽  
Christopher D. BYRNE
Keyword(s):  
Reverse Transcriptase ◽  
Copy Number ◽  
Accurate Determination ◽  
Fold Increase ◽  
Pcr Primers ◽  
Cdna Synthesis ◽  
Reverse Transcriptase Pcr ◽  
Target Mrna ◽  
Mrna Copy Number ◽  
Synthesis Reactions

Quantitative competitive reverse-transcriptase PCR is the most sensitive method for studying gene expression. To investigate whether the accuracy of the calculated target mRNA copy number is affected by the cDNA priming process, we utilized primers of different lengths, concentrations and primer sequences to prime cDNA synthesis reactions. Our results show a ≈ 19-fold increase in the calculated mRNA copy number from cDNA synthesis reactions primed with random hexamers (P< 0.001, n = 4), and a ≈ 4-fold increase in copy number with a specific hexamer (P< 0.001, n = 4) compared with that obtained with a 22-mer-sequence-specific primer. The increase in calculated mRNA copy number obtained by priming cDNA synthesis with the shorter specific and non-specific primers could be explained largely by the synthesis of truncated standard cDNA molecules lacking a requisite binding site for amplification with PCR primers. Since these truncated standard cDNA molecules could not be amplified and standard RNA is used to quantify target mRNA copy number, this phenomenon resulted in overestimation of target mRNA copy number. In conclusion, accurate determination of target mRNA copy number is most likely if a long specific antisense primer is used to prime cDNA synthesis.

scholarly journals Immunomagnetic Separation Combined with Real-Time Reverse Transcriptase PCR Assays for Detection of Norovirus in Contaminated Food

2008 ◽  
Vol 74 (13) ◽  
pp. 4226-4230 ◽  
Author(s):  
YoungBin Park ◽  
You-Hee Cho ◽  
YoungMee Jee ◽  
GwangPyo Ko
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Immunomagnetic Separation ◽  
Rt Pcr ◽  
Reverse Transcriptase Pcr ◽  
Food Borne ◽  
Contaminated Food ◽  
Microbial Contaminants ◽  
Pcr Assays ◽  
Sensitive Method

ABSTRACT We developed an immunomagnetic separation (IMS) technique combined with real-time TaqMan reverse transcriptase PCR (RT-PCR), which allowed detection of norovirus at a level as low as 3 to 7 RT-PCR units from artificially contaminated strawberries. The inoculum recovery rate ranged from 14 to 30%. The data demonstrate that IMS combined with real-time RT-PCR will be useful as a rapid and sensitive method for detecting food-borne microbial contaminants.

scholarly journals Comparison of Enzyme Immunoassay and Reverse Transcriptase PCR for Identification of Serotype G9 Rotaviruses

1999 ◽  
Vol 37 (10) ◽  
pp. 3187-3193 ◽  
Author(s):  
Barbara S. Coulson ◽  
Jon R. Gentsch ◽  
Bimal K. Das ◽  
M. K. Bhan ◽  
Roger I. Glass
Keyword(s):  
Reverse Transcriptase ◽  
Enzyme Immunoassay ◽  
High Titer ◽  
New Delhi ◽  
Rt Pcr ◽  
Freeze Thaw ◽  
Reverse Transcriptase Pcr ◽  
Pcr Genotyping ◽  
Sensitive Method

While only four globally important rotavirus G serotypes (1 to 4) have been documented, many studies suggest that serotype G9 viruses may be widely distributed and more important than previously recognized. We have evaluated 10 serotype G9 rotavirus-neutralizing monoclonal antibodies (MAbs) directed to VP7, which bound by direct enzyme immunoassay (EIA) to P1A[8], G9 rotaviruses F45, WI61, and AU32, for their ability to recognize the New Delhi G9 rotavirus 116E. Only one MAb (MAb F45:1) bound to P[11], G9 virus 116E to a high titer by EIA. This MAb was incorporated into an indirect EIA for G serotyping, which was validated with prototype cultivable human rotaviruses of G types 1 to 4 and 9. The EIA was compared with genotyping by reverse transcriptase PCR (RT-PCR) under code for the determination of the G types of rotaviruses obtained from neonates in New Delhi, India. The sensitivities of RT-PCR and EIA (after two additional freeze-thaw cycles) for the typing of G9 rotaviruses were 91 and 86%, respectively, for 24 culture-adapted rotavirus strains. The untypeable culture-adapted rotavirus samples also were unreactive with VP7 group antigen-reactive MAb 60. After two additional freeze-thaw cycles, only 26 of 42 (62%) of stools containing rotavirus typed as G9 by RT-PCR were positive for G9 rotavirus by EIA. Stools containing rotavirus untypeable by EIA contained significantly less MAb 60-reactive VP7 antigen (P = 0.0001) than the stools containing typeable rotavirus. Thus, RT-PCR genotyping was the more sensitive method for determination of G9 type, but a serotype was readily determined in rotavirus samples containing MAb 60-reactive VP7 antigen by an EIA that incorporates MAb F45:1.

A Reverse Transcriptase PCR Technique for the Detection and Viability Assessment of Kluyveromyces marxianus in Yoghurt

2006 ◽  
Vol 69 (9) ◽  
pp. 2210-2216 ◽  
Author(s):  
MARÍA BELÉN MAYORAL ◽  
ROSARIO MARTÍN ◽  
PABLO E. HERNÁNDEZ ◽  
ISABEL GONZÁLEZ ◽  
TERESA GARCÍA
Keyword(s):  
Reverse Transcriptase ◽  
Kluyveromyces Marxianus ◽  
18S Rrna ◽  
Yeast Cells ◽  
Rt Pcr ◽  
Viability Assessment ◽  
Reverse Transcriptase Pcr ◽  
Low Concentrations ◽  
Pcr Method ◽  
High Level

A fast and sensitive reverse transcriptase PCR (RT-PCR) method was developed for the detection of viable Kluyveromyces marxianus in yoghurt. Yeast-specific primers were used with the RT-PCR to evaluate the suitability of 18S rRNA as a target for the detection of viable yeasts in pure culture and yoghurt. The RT-PCR assay was able to detect down to 102 CFU ml−1 in yoghurt samples contaminated with viable yeast cells. Application of the RT-PCR method to commercial yoghurt samples demonstrated the utility of this technique for detection of low concentrations of viable yeast cells in naturally contaminated dairy products. The 18S rRNA molecule is an appropriate target for cell viability assessment because of its limited persistence after cell death and the resultant high level of sensitivity of the assay.

Improvements in the RT-PCR detection of enteric viruses in environmental samples

1998 ◽  
Vol 38 (12) ◽  
pp. 83-86 ◽  
Author(s):  
K. J. Schwab ◽  
F. H. Neill ◽  
M. K. Estes ◽  
R. L. Atmar
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcriptase ◽  
Environmental Samples ◽  
Internal Standard ◽  
Pcr Amplification ◽  
Enteric Viruses ◽  
New Method ◽  
Rt Pcr ◽  
Pcr Product ◽  
Enzyme Method

Current methods for the detection of nucleic acid from enteric viruses in environmental samples usually involve extensive concentration and purification of target viruses followed by RT-PCR amplification using two enzymes, reverse transcriptase and Taq polymerase. We have developed a modified method that improves RT-PCR assays by: (i) the use of an RT-PCR internal standard control RNA to identify potential false negative results caused by inhibition of RT-PCR enzymes; (ii) the use of rTth (Perkin-Elmer, Foster City, CA), a heat-stable enzyme that functions as both a reverse transcriptase and DNA polymerase in a single-tube, single-buffer, elevated-temperature reaction; and (iii) the use of thermolabile uracil N-glycosylase (HK-UNG) (Epicentre Technologies, Madison, WI) to prevent PCR product carryover contamination. The new method was compared to the traditional two-enzyme, RT-PCR method for detection of Norwalk virus (NV) and hepatitis A virus (HAV) in buffer, stool, clam and oyster samples. The new method was at least as sensitive in NV and HAV detection compared to the traditional two-enzyme method. The internal standard control successfully detected inhibitors to RT-PCR amplification. NV and HAV PCR products generated with dUTP replacing dTTP during amplification were seeded into subsequent samples to test the prevention of PCR product carryover contamination by HK-UNG. The new method successfully eliminated PCR product carryover contamination in contrast to the traditional two-enzyme method. These improvements to viral nucleic acid detection have the potential to improve sensitivity, specificity and confidence in RT-PCR results.

scholarly journals Detection of Mycobacterium tuberculosis Group Organisms in Human and Mouse Joint Tissue by Reverse Transcriptase PCR: Prevalence in Diseased Synovial Tissue Suggests Lack of Specific Association with Rheumatoid Arthritis

2001 ◽  
Vol 69 (3) ◽  
pp. 1821-1831 ◽  
Author(s):  
Karen E. Kempsell ◽  
Charles J. Cox ◽  
Andrew A. McColm ◽  
Julie A. Bagshaw ◽  
Richard Reece ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Mycobacterium Tuberculosis ◽  
Synovial Fluid ◽  
Reverse Transcriptase ◽  
Synovial Tissue ◽  
Rt Pcr ◽  
Diverse Range ◽  
Susceptible Host ◽  
Joint Tissue ◽  
Reverse Transcriptase Pcr

ABSTRACT Infection with mycobacterial species, including Mycobacterium tuberculosis, has long been implicated in the etiopathology of rheumatoid arthritis (RA) on the basis of clinical and pathological similarities between tuberculosis and RA. Despite evidence of immune responses to mycobacterial antigens in RA patient synovial fluid, cross-reactivity between these and host joint antigens, and the presence of M. tuberculosis protein antigen in RA synovial fluid, a definite causal association with RA has not been shown. Previous studies from our laboratory using reverse transcriptase PCR (RT-PCR) of bacterial rRNAs have shown RA synovium to be colonized by a diverse range of bacteria, most of commensal origin. However, M. tuberculosis group organism (MTG) RNA sequences were found in one RA patient tissue. Since this was considered of sufficient interest to warrant further investigation, we devised a M. tuberculosis-specific nested RT-PCR test which could be used for detection of MTG in a mixed pool of bacterial crDNAs. This test was used to investigate the distribution of MTG in RA synovial tissue and also non-RA arthritis and healthy control tissues and was also used to examine the tissue distribution of MTG in an acute and chronic model ofM. tuberculosis infection in the BALB/c mouse. MTG sequences were found in a high proportion of RA patient synovial tissues but also in non-RA arthritis control tissues at lower frequency. This likely reflects trafficking of persistent M. bovis BCG to inflamed joint tissue, irrespective of cause. MTG were not found in healthy synovial tissue or the tissue of patients with undifferentiated arthritis. In both the acute and chronic models of infection in BALB/c mice, M. tuberculosis was also found to have trafficked to joint tissues, however, no signs of inflammation, arthritis, or pathology associated with M. tuberculosisinfection was seen. These combined results would argue against a specific causal role of MTG in RA-like arthritis; however, their role as adjuvant in immune dysfunction in an innately susceptible host cannot be excluded.

scholarly journals Use of Reverse Transcriptase PCR in Early Diagnosis of Rift Valley Fever

2002 ◽  
Vol 9 (3) ◽  
pp. 713-715 ◽  
Author(s):  
A. A. Sall ◽  
E. A. Macondo ◽  
O. K. Sène ◽  
M. Diagne ◽  
R. Sylla ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Rift Valley ◽  
Rift Valley Fever ◽  
Virus Isolation ◽  
Immunoglobulin M ◽  
Antibody Detection ◽  
Rt Pcr ◽  
Valley Fever ◽  
Reverse Transcriptase Pcr ◽  
Sensitive Tool

ABSTRACT Reverse transcriptase PCR (RT-PCR) for diagnosis of Rift Valley fever (RVF) was evaluated by using 293 human and animal sera sampled during an RVF outbreak in Mauritania in 1998. Results of the RT-PCR diagnostic method were compared with those of virus isolation (VI) and detection of immunoglobulin M (IgM) antibodies. Our results showed that RT-PCR is a specific, sensitive tool for RVF diagnosis in the early phase of the disease and that its results do not differ significantly from those obtained by VI. Moreover, the combined results of RT-PCR and IgM antibody detection were in 100% concordance with the results of VI.

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