Simple Quantitative Reverse Transcribed-Polymerase Chain Reaction (RT-PCR) Method Involving Recombinant RNA Generated by a False-Priming PCR Product

1994 ◽  
Vol 116 (6) ◽  
pp. 1198-1201 ◽  
Author(s):  
Makoto Sato ◽  
Mutsuhiko Mizobuchi ◽  
Koji Murao ◽  
Miho Tamaki ◽  
Jiro Takahara
Keyword(s):  
Polymerase Chain Reaction ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Product ◽  
Pcr Method ◽  
Polymerase Chain

scholarly journals Deteksi Plasmodium falciparum dengan menggunakan metode real-time polymerase chain reaction di daerah Likupang dan Bitung

2016 ◽  
Vol 4 (1) ◽  
Author(s):  
David C. Tooy ◽  
Janno B. Bernadus ◽  
Angle Sorisi
Keyword(s):  
Polymerase Chain Reaction ◽  
Plasmodium Falciparum ◽  
Real Time ◽  
Real Time Pcr ◽  
18S Rrna ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Negative Results ◽  
Pcr Method ◽  
Polymerase Chain

Abstract: Malaria is one of the most important parasitic disease which is caused by Plasmodium spp. There are approximately 1,2 billion people in the world with high risk of getting malaria. Plasmodium falciparum (P. falciparum) is the cause of tropical malaria or falciparum malaria, and is responsible for most of the mortality rate. Currently, real-time polymerase chain reaction (RT-PCR) is being studied as an alterative of conventional malarian examination. Mangold et al reported that RT-PCR have 94.1% sensitivity and 100% specificity compared to microscopic examination in detecting P. falciparum. The aim of this research is to detect the presence of P. falciparum using RT-PCR in Likupang and Bitung region. This research were using descriptive design to find out the capability of real-time PCR method to detect P. falciparum in Likupang dan Bitung region. The researcher have examined 71 samples which are fulfill the research sample’s criteria. Postive results of P. falciparum found in 18 samples (25,3%) and negative results in 53 samples (74,6%) of total 71 samples with using RT-PCR. No positive results were found in samples from Likupang. There are positive result of P. falciparum in samples from Bitung. It is concluded that RT-PCR method can detect the presence of P. falciparum from the samples obtained from Likupang and Bitung based on the presence of its DNA. This detection efford is done by using 18S rRNA as target gene and ajust specific temperature on the RT-PCR instrument.Keywords: Plasmodium falciparum, Real-time Polymerase Chain Reaction (PCR), DetectionAbstrak: Malaria merupakan salah satu penyakit penting yang disebabkan oleh parasit Plasmodium spp. Kira-kira 1,2 miliar penduduk dunia memiliki risiko tinggi untuk mendapat malaria. Di Indonesia sendiri, terdapat 343.527 kasus terkonfirmasi dan 45 kematian karena malaria. Plasmodium falciparum (P. Falciparum) merupakan penyebab dari malaria tropika atau malaria falsiparum, dan bertanggung jawab atas sebagian besar angka mortalitas. Saat ini Real-Time Polymerase Chain Reaction (RT-PCR) telah banyak diteliti sebagai alternatif dari pemeriksaan malaria. Mangold dkk melaporkan bahwa real-time PCR memiliki nilai sensitivitas 94,1% dan nilai spesifisitas 100% terhadap pemeriksaan mikroskopis dalam mendeteksi P. falciparum. Penelitian bertujuan untuk mendeteksi P. falciparum dengan menggunakan RT-PCR di daerah Likupang dan Bitung. Penelitian ini menggunakan rancangan penelitian deskriptif untuk mengetahui kemampuan metode real-time PCR dalam mendeteksi P. falciparum di daerah Likupang dan Bitung. Tujuan penelitian ini ialah untuk mendeteksi keberadaan P. falciparum dengan menggunakan metode real-time PCR di daerah Likupang dan Bitung. Peneliti memeriksa 71 sampel darah yang memenuhi kriteria sampel penelitian. Hasil positif P. falciparum ditemukan pada 18 sampel (25,3 %) dan hasil negatif pada 53 sampel (74,6 %) dari total 71 sampel dengan menggunakan RT-PCR. Tidak ditemukannya hasil positif P. falciparum pada sampel dari Likupang. Ditemukan hasil positif P. falciparum pada sampel dari Bitung. Simpulan: Metode RT-PCR dapat mendeteksi P. falciparum berdasarkan keberadaan DNA-nya pada sampel yang diperoleh dari daerah Likupang dan Bitung. Deteksi ini berhasil dilakukan dengan menggunakan 18S rRNA sebagai gen target dan pengaturan suhu tertentu pada instrument RT-PCR.Kata kunci: P. falciparum, Real-time Polymerase Chain Reaction (PCR), Detection

scholarly journals Species-Specific Polymerase Chain Reaction Amplification of Camel (Camelus) DNA Extracts

2005 ◽  
Vol 88 (5) ◽  
pp. 1394-1398 ◽  
Author(s):  
Ying Chen ◽  
Ya-Jun Wu ◽  
Bao-Liang Xu ◽  
Jing Wan ◽  
Zeng-Ming Qian
Keyword(s):  
Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Amplification ◽  
Chain Reaction ◽  
Pcr Product ◽  
Mitochondrial Cytochrome B ◽  
Base Pair Fragment ◽  
Sensitive Polymerase Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Species Specific

Abstract A sensitive polymerase chain reaction (PCR) method based on amplification of a specific DNA fragment was established for the identification of camel (Camelus) materials. The species-specific primer pair L183/H372 was designed based on the nucleotide sequence of the mitochondrial cytochrome b gene, and its specificity was confirmed by amplification of 3 camel (domestic double-humped camel, wild double-humped camel, wild one-humped camel) samples and 11 non-Camelus animal (sheep, goat, pig, chicken, cattle, fish, dog, horse, donkey, deer, and rabbit) materials. An expected 208 base pair fragment was amplified from camel materials; no cross-reactive or additional fragments were generated from other animal materials. Taq I restriction endonuclease digestion of the unpurified PCR product can be used routinely to confirm the camel origin of the amplified sequence.

scholarly journals Inhibitory AKT2 And Vegr2 Genes Expression of Avicennia Marina Using RT-PCR On Widr Cells

2020 ◽  
Vol 8 (1) ◽  
pp. 1-4
Author(s):  
Qurrohman, T ◽  
Hasibuan, P.A.Z. ◽  
Basyuni, M
Keyword(s):  
Polymerase Chain Reaction ◽  
Cancer Chemoprevention ◽  
Avicennia Marina ◽  
Hexane Extract ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Genes Expression ◽  
Regulated Expression ◽  
Pcr Method ◽  
Polymerase Chain

Objectives: To evaluate the activities the anticancer of n-hexane extract of Avicennia marina leaves on WiDr cells in down-regulated of expression of Akt2 and VEGFR2 genes. Methods: Avicennia marina leaves  were dried and extracted with n-hexane, analyzed the down-regulated Akt 2 and VEGFR2 expression which was determined the Reverse Transcription Polymerase Chain Reaction (RT-PCR) method. Results:N-hexane extract of Avicennia marina, in which there were down regulated expression Akt 2 and VEGFR2 of 0.43 and 0.50 WiDr cells. N-hexane extract of mangrove leaves has cancer chemoprevention activity with down-regulated on WiDr cells. Conclusions: :N-hexane extract of Avicennia marina leaves had anticancer activity with down-regulated on WiDr cells, suggest that significantly inhibit the expression of Akt2 and VEGFR2 genes.    

Detection of Enteroviral RNA Sequences in Different Water Samples

1993 ◽  
Vol 27 (3-4) ◽  
pp. 219-222 ◽  
Author(s):  
D. Tougianidou ◽  
K. Botzenhart
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Water Samples ◽  
Test System ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Rna Sequences ◽  
Pcr Product ◽  
Filter Unit ◽  
Polymerase Chain

Viruses were isolated from different water samples by Sterivex-Filtration. The nucleic acids were isolated in the filter unit and purified by phenol-chloroform extraction and ethanol precipitation. Reverse transcription and polymerase chain reaction (RT/PCR) were performed with primer pairs complementary to sequences of the enteroviral 5’ noncoding region. Amplified sequences were detected by hybridisation with an oligonucleotide complementary to a part of the PCR product. The test system seems to be sensitive and specific In the detection of enteroviral RNA.

scholarly journals Comparison of Reverse Transcription-Polymerase Chain Reaction, Immunohistochemistry, and Fluorescence In Situ Hybridization Methodologies for Detection of Echinoderm Microtubule-Associated Proteinlike 4–Anaplastic Lymphoma Kinase Fusion–Positive Non–Small Cell Lung Carcinoma: Implications for Optimal Clinical Testing

2012 ◽  
Vol 136 (7) ◽  
pp. 796-803 ◽  
Author(s):  
Michelle L. Wallander ◽  
Katherine B. Geiersbach ◽  
Sheryl R. Tripp ◽  
Lester J. Layfield
Keyword(s):  
Polymerase Chain Reaction ◽  
Small Cell ◽  
Wild Type ◽  
Rt Pcr ◽  
Small Cell Lung ◽  
Chain Reaction ◽  
Anaplastic Lymphoma ◽  
Pcr Method ◽  
Polymerase Chain

Context.—Echinoderm microtubule–associated proteinlike 4–anaplastic lymphoma kinase (EML4-ALK) gene fusions are detected in 3% to 13% of non–small cell lung carcinomas. Accurate testing for detection of EML4-ALK fusions is essential for appropriate therapy selection. Objective.—To compare reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and fluorescence in situ hybridization (FISH) methodologies for detection of EML4-ALK fusions. Design.—Forty-six pulmonary adenocarcinomas were selected with enrichment for wild-type epidermal growth factor receptor (EGFR) status (wild type, n  =  42; mutant, n  =  4). Specimens were tested by IHC (Dako; clone ALK1), FISH (Abbott Molecular; LSI ALK break apart), and RT-PCR (variants 1 and 3a/b). Results.—EML4-ALK variant 3a/b was detectable by RT-PCR, FISH, and IHC in 4% (2 of 46) of specimens. Complete agreement among FISH and IHC reviewers was obtained for variant 3a/b. No concordance existed among methodologies for the detection of EML4-ALK variant 1. The RT-PCR method detected variant 1 in 20% (9 of 46) of specimens. Agreement among FISH viewers was poor for variant 1 because only 11% (1/9) of specimens were scored as positive by all 3 viewers. The sensitivity of IHC for detection of variant 1 was also poor because only 1 of 9 samples (11%) was scored as positive. Overall, the frequency of EML4-ALK variants 1 and 3a/b was 24% (11 of 46) in adenocarcinomas enriched for wild-type EGFR status. One EML4-ALK variant 1 fusion was found to coexist with an EGFR exon 21 mutation. Conclusions.—The FISH interpretation demonstrated great variability among observers. The RT-PCR method was the most sensitive and least-subjective methodology for detection of EML4-ALK fusions.

scholarly journals METHOD FOR FMD VIRUS 146S COMPONENT CONCENTRATION DETERMINATION WITH REAL-TIME REVERSE TRANSCRIPTION – POLYMERASE CHAIN REACTION IN VACCINE RAW MATERIALS

2018 ◽  
pp. 31-35
Author(s):  
M. I. Doronin ◽  
A. M. Timina ◽  
D. A. Lozovoy ◽  
V. A. Starikov ◽  
D. V. Mikhalishin ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Regression Model ◽  
Real Time ◽  
Reverse Transcription ◽  
Raw Materials ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain

Method for determination of foot-and-mouth disease (FMD) virus 146S component concentration with real-time reverse transcription – polymerase chain reaction (rtRT-PCR) in vaccine raw materials is developed. Negative regression model of  С146S = (30.269 – Ct)/4.155 type allowing determination of FMDV 146S particle concentrations based on the amplification threshold cycle values (Ct) is proposed. It has been experimentally proven that the quantity of the 146S component determined by the real-time RT-PCR method using developed regression model and contained in the inoculation dose of FMD vaccine confers protection to the vaccinated animals against generalized FMD of A, O, Asia-1 types. rtRT-PCR method is highly sensitive and allows rapid and highly reliable estimation of  the 146S antigen concentration in FMD vaccine. The method for 146S particle quantity determination by real-time RT-PCR using the regression model is reliable and demonstrates high correlation (95.5–99.0%) with the complement fixation test results.

scholarly journals Noninvasive prenatal screening test for compound heterozygous beta thalassemia using an amplification refractory mutation system real-time polymerase chain reaction technique

2019 ◽  
Vol 11 (3) ◽  
Author(s):  
Narutchala Suwannakhon ◽  
Tanapat Pangeson ◽  
Teerapat Seeratanachot ◽  
Khwanruedee Mahingsa ◽  
Arunee Pingyod ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Beta Thalassemia ◽  
Amplification Refractory Mutation System ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Mutation System ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Thalassemia Mutation

We propose using a modified amplification refractory mutation system real-time polymerase chain reaction (ARMS RTPCR) technique to exclude the invasive prenatal diagnosis for a non-paternally inherited beta thalassemia mutation in couples atrisk for having a baby with CHBT. The ARMS RT-PCR method was performed for 36 at-risk couples by using isolated fetal cell-free DNA from maternal plasma. The modified ARMS RT-PCR primers targeted one of the following paternally inherited beta thalassemia mutation: -28 A→G, CD17 A→T, CD 26 G→A, IVS1-1 G→T and CD 41-42 -CTTT. The method could be successfully employed for NIPST starting with the 7th week of gestation. The results showed that 19 pregnant women were negative for PIBTM (53%). After an on-track and on-time of one year, including postnatal thalassemia blood tests, none of the babies showed symptoms or signs of beta thalassemia disease. We concluded that the modified ARMS RT-PCR method was an accurate, cost-effective and feasible method for use as a NIPST for at-risk couples with the potential of having a baby with CHBT.

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