Reference virus as an internal standard for Semliki forest virus real-time PCR quantification

It is considered that GGTA1 knockout (KO) pig production via somatic cell NT would overcome the problem of immune rejection after xenotransplantation. It is reported that although GGTA KO mice showed only a mild increase in sialyltransferase gene expression, GGTA1 deficiency in pig could increase the sialyltransferase activities, non-Gal epitope expression, consequently may raise non-Gal xenoantigenicity. Therefore, in the present study we investigated whether the expression level of Sia-containing glycoconjugate mRNA in transgenic pigs could be affected by knocking out the GGTA1 gene. Besides GGTA1 KO pigs, double genes overexpressing pigs (2TG) and GGTA1 KO with double genes overexpressing (KO+2TG) pigs were produced by somatic cell NT. For the present study, fibroblasts were isolated from wild-type pigs without gene modification, 2TG, GGTA1 KO, and KO+2TG pig. The GAPDH gene was used as an internal standard to normalise the real-time PCR (RT-qPCR) analysis reaction efficiency and to quantify mRNA in pigs-derived fibroblast. The expression levels were compared between them (RT-qPCR) in triplicate for each sample. Oligonucleotide primers for real-time PCR were designed for Hanganutziu-Deicher antigen (ST3Gal1–4, ST6Gal1) and Sialyl-Tn antigen (ST6GalNac1, ST6GalNac2, and ST6GalNac6) analysis. For statistical analysis, one-way ANOVA with Dunn’s multiple comparison test were used. The mRNA expression of GGTA1 KO and KO+2TG pig derived fibroblasts cells genes showed that ST3Gal1, ST3Gal2, ST3Gal3, and ST6Gal1 gene expression were significantly up-regulated compared to the wild and 2TG pigs (P < 0.05). However, ST3Gal4, Sialyl-Tn antigen including ST6GalNac1, ST6GalNac2, and ST6GalNac6 in KO+2TG pigs were not different compared with the wild pigs (P > 0.05), whereas only GGTA1 KO pigs showed significantly higher expressions than wild, 2TG, and KO+2TG pigs (P < 0.05). These results demonstrated that GGTA KO pig-derived cells exhibit a higher Hanganutziu-Deicher antigen on glycoprotein and glycolipid than controls, and KO+2TG pig exhibit no differences when compared with GGTA1 KO pig, indicating that they do not act as an immune antigen in xenograft. Overall, the increase in glycosyltransferase expression suggests a corresponding increase in the cell surface sialyation in GGTA KO pig cells. For xenotransplantation, KO+2TG pigs were more preferable because of absence of immune rejection for Sia-containing glycoconjugate on glycoprotein and glycolipid than GGTA KO pigs. This study was supported by the Ministry of Trade, Industry and Energy (#10048948), Korea IPET (#114059–3), Research Institute for Veterinary Science, TS Corporation, and the BK21 plus program.

Download Full-text

Development and Evaluation of a Duplex Real-Time PCR Assay With a Novel Internal Standard for Precise Quantification of Plasma DNA

Annals of Laboratory Medicine ◽

10.3343/alm.2017.37.1.18 ◽

2017 ◽

Vol 37 (1) ◽

pp. 18-27 ◽

Cited By ~ 4

Author(s):

Dan Chen ◽

Shiyang Pan ◽

Erfu Xie ◽

Li Gao ◽

Huaguo Xu ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Internal Standard ◽

Plasma Dna ◽

Pcr Assay ◽

Precise Quantification

Download Full-text

Development of a Real-Time TaqMan PCR Method for Absolute Quantification of the Biocontrol Agent Esteya vermicola

Plant Disease ◽

10.1094/pdis-10-19-2076-re ◽

2020 ◽

Vol 104 (6) ◽

pp. 1694-1700

Author(s):

Hai-Hua Wang ◽

Can Yin ◽

Jie Gao ◽

Ran Tao ◽

Chun-Yan Wang ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Biocontrol Agent ◽

Internal Standard ◽

Absolute Quantification ◽

Pinewood Nematode ◽

Hyphal Length ◽

Taqman Pcr ◽

Esteya Vermicola ◽

The Absolute

Esteya vermicola has been used as an effective biocontrol agent for the management of the pinewood nematode, Bursaphelenchus xylophilus. Tools for monitoring the colonization and parasitism patterns of E. vermicola are required for the development of highly effective biocontrol strategies. Because the TaqMan PCR technique is effective for quantification of species in environmental samples, a real-time PCR-based methodology was developed for absolute quantification of E. vermicola via internal standard addition and extrapolation of DNA quantity to hyphal length. Primers and a probe for the 28S ribosomal RNA gene of E. vermicola were designed, and nested TaqMan real-time PCR-based quantification was performed. In addition, internal standard-based yield measurement was correlated to the absolute quantity of target genomic DNA. Moreover, an extrapolation curve obtained by optical microscopy and image analysis of the mycelia was constructed for the measurement of fungal hyphal length. The absolute quantification method developed in the present study provides a sensitive and accurate technique to quantify fungal density in either wood or other substrate samples and can be used as an effective tool for future studies of biocontrol agents.

Download Full-text

Development of a Real-Time PCR Assay for Identification of Coccidioides immitis by Use of the BD Max System: TABLE 1

Journal of Clinical Microbiology ◽

10.1128/jcm.02731-14 ◽

2015 ◽

Vol 53 (3) ◽

pp. 926-929 ◽

Cited By ~ 13

Author(s):

Marilyn Mitchell ◽

Dominic Dizon ◽

Robert Libke ◽

Michael Peterson ◽

David Slater ◽

...

Keyword(s):

Sample Preparation ◽

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Hospital Setting ◽

Internal Standard ◽

Specimen Preparation ◽

Coccidioides Immitis ◽

Rt Pcr ◽

Content Type

Rapid real-time PCR (RT-PCR) can be performed in a community hospital setting to identifyCoccidioidesspecies using the new Becton Dickinson molecular instrument BD Max. Following sample preparation, DNA extraction and PCR were performed on the BD Max using the BD Max extraction kit ExK-DNA-1 test strip and a master mix prepared by BioGX (Birmingham, AL). Sample preparation took 2 h, and testing on the BD Max took an additional 2 h. Method sensitivity and specificity were evaluated along with the limits of detection to confirm that this convenient method would provide medically useful information. Using serial dilutions, the lower limit of detection was determined to be 1 CFU/μl. Testing with this method was validated using samples from various body sites, including bronchial alveolar lavage (BAL) fluid; sputum and lung tissue samples; and pleural and spinal fluids. Safety protocols were established, and specimen preparation processes were developed for the various types of specimens. The range for the cycle threshold (CT) indicating adequate fluorescent signal to signify a positive result was established along with the acceptable range for the internal standard. Positive controls run with each batch were prepared by spiking a pooled BAL fluid specimen with a known dilution ofCoccidioides immitisorganism. Our experience with testing >330 patient samples shows that clinically relevant information can be available within 4 h using an RT-PCR method on the BD Max to identifyCoccidioidesspp., with sensitivity equivalent to culture.

Download Full-text

Mediator Probe PCR: A Novel Approach for Detection of Real-Time PCR Based on Label-Free Primary Probes and Standardized Secondary Universal Fluorogenic Reporters

Clinical Chemistry ◽

10.1373/clinchem.2012.186734 ◽

2012 ◽

Vol 58 (11) ◽

pp. 1546-1556 ◽

Cited By ~ 15

Author(s):

Bernd Faltin ◽

Simon Wadle ◽

Günter Roth ◽

Roland Zengerle ◽

Felix von Stetten

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Homo Sapiens ◽

Internal Standard ◽

Pcr Amplification ◽

Nuclease Activity ◽

Target Sequence ◽

Label Free ◽

Hydrolysis Probe ◽

Detection Techniques

BACKGROUND The majority of established techniques for monitoring real-time PCR amplification involve individual target-specific fluorogenic probes. For analysis of numerous different targets the synthesis of these probes contributes to the overall cost during assay development. Sequence-dependent universal detection techniques overcome this drawback but are prone to detection of unspecific amplification products. We developed the mediator probe PCR as a solution to these problems. METHODS A set of label-free sequence-specific primary probes (mediator probes), each comprising a target-specific region and a standardized mediator tag, is cleaved upon annealing to its target sequence by the polymerases' 5′ nuclease activity. Release of a mediator triggers signal generation by cleavage of a complementary fluorogenic reporter probe. RESULTS Real-time PCR amplification of human papillomavirus 18 (HPV18), Staphylococcus aureus, Escherichia coli, and Homo sapiens DNA dilution series showed exceptional linearity when detected either by novel mediator probes (r2 = 0.991–0.999) or state-of-the-art hydrolysis probes (TaqMan probes) (r2 = 0.975–0.993). For amplification of HPV18 DNA the limits of detection were 78.3 and 85.1 copies per 10-μL reaction when analyzed with the mediator probe and hydrolysis probe, respectively. Duplex amplification of HPV18 target DNA and internal standard had no effects on back calculation of target copy numbers when quantified with either the mediator probe PCR (r2 = 0.998) or the hydrolysis probe PCR (r2 = 0.988). CONCLUSIONS The mediator probe PCR has equal performance to hydrolysis probe PCR and has reduced costs because of the use of universal fluorogenic reporters.

Download Full-text

Determination of unprocessed genetically modified soybean in foods using simplex and duplex real-time PCR with an internal standard

International Journal of Food Science & Technology ◽

10.1111/j.1365-2621.2009.01996.x ◽

2009 ◽

Vol 44 (9) ◽

pp. 1778-1785 ◽

Cited By ~ 2

Author(s):

Naoki Harikai ◽

Shin Saito ◽

Atsuko Tanaka ◽

Kenji Kinoshita

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Genetically Modified ◽

Internal Standard ◽

Genetically Modified Soybean

Download Full-text

A Novel PCR Method for Quantification of Buckwheat by Using a Unique Internal Standard Material

Journal of Food Protection ◽

10.4315/0362-028x-69.10.2478 ◽

2006 ◽

Vol 69 (10) ◽

pp. 2478-2486 ◽

Cited By ~ 19

Author(s):

TAKASHI HIRAO ◽

MASAYUKI HIRAMOTO ◽

SHINSUKE IMAI ◽

HISANORI KATO

Keyword(s):

Real Time ◽

Dna Extraction ◽

Food Allergen ◽

Real Time Pcr ◽

Internal Standard ◽

Food Samples ◽

Internal Transcribed Spacer Region ◽

Standard Material ◽

Wild Buckwheat ◽

Pcr Targeting

A novel quantitative and specific method for detection of buckwheat, a known food allergen, in diverse food materials was developed by using a unique internal standard to compensate for the variability in DNA extraction and amplification efficiencies. The method was based on a real-time PCR targeting the internal transcribed spacer region of Fagopyrum spp. and was designed to detect both cultivated and wild buckwheat, because wild buckwheat might be potentially allergenic. As the internal standard material, ground seeds of statice (Limonium sinuatum) were added to food samples prior to DNA extraction, and the amount of statice DNA measured by real-time PCR was used to standardize the buckwheat content. Statice, an ornamental plant, was chosen as the internal standard material because it was readily available and was inferred to be least likely to be commingled in foods. The specificity of the PCR system was tested against commonly used food materials of plant origin. Quantitative results expressed in buckwheat protein concentrations (mean ± standard deviation) for various food samples prepared to contain 10 ppm (wt/wt) of buckwheat flour (corresponding to 1.2-μg/g [ppm] buckwheat protein) ranged from 0.7 ± 0.2 (rice) to 0.9 ± 0.4 (wheat) and for 100-ppm (wt/wt) samples (12-μg/g [ppm] buckwheat protein) from 7.7 ± 1.0 (pepper) to 9.8 ± 0.5 (wheat) μg/g (ppm). The method's accuracy, sensitivity, and specificity were considered sufficient for detection of buckwheat contamination at the level required for compliance with the Japanese Food Allergen Labeling Regulation.

Download Full-text