scholarly journals Quantitative analysis of the dystrophin gene by real-time PCR

2012 ◽  
Vol 64 (2) ◽  
pp. 787-792 ◽  
Author(s):  
Nela Maksimovic ◽  
Ana Andjelkovic ◽  
Vedrana Milic-Rasic ◽  
Vidosava Rakocevic-Stojanovic ◽  
Biljana Kastratovic-Kotlica ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Gene Dosage ◽  
Neuromuscular Disorders ◽  
Cost Effective ◽  
Dystrophin Gene ◽  
Becker Muscular Dystrophy ◽  
Carrier Status ◽  
Gene Deletions ◽  
Pcr Method

Duchenne and Becker muscular dystrophy (DMD/BMD) are severe X-linked neuromuscular disorders caused by mutations in the dystrophin gene. Our aim was to optimize a quantitative real-time PCR method based on SYBR? Green I chemistry for routine diagnostics of DMD/BMD deletion carriers. Twenty female relatives of DMD/BMD patients with previously detected partial gene deletions were studied. The relative quantity of the target exons was calculated by a comparative threshold cycle method (??Ct). The carrier status of all subjects was successfully determined. The gene dosage ratio for non-carriers was 1.07?0.20, and for carriers 0.56?0.11. This assay proved to be simple, rapid, reliable and cost-effective.

Fluorescent PCR Based Gene Dose Assessment for Detection of Deletion Mutations in the FIX Gene Among Carriers of Hemophilia B.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3486-3486
Author(s):  
Eunice Sindhuvi Edison ◽  
G. Sankari Devi ◽  
G. Jayandharan ◽  
Shaji R Velayudhan ◽  
Auro Viswabandya ◽  
...  
Keyword(s):  
Linkage Analysis ◽  
Real Time ◽  
Real Time Pcr ◽  
Gene Dosage ◽  
Family Members ◽  
Peak Height ◽  
Hemophilia B ◽  
Carrier Status ◽  
Fluorescent Pcr ◽  
The Family

Abstract Abstract 3486 Poster Board III-423 Haemophilia B (HB), an X linked inherited disorder is caused by heterogeneous mutations in the F9 gene. Approximately 3% of hemophilia B patients have major deletions in the F9 gene. Gross and small deletions in the F9 gene in HB affected males are easily detected by PCR but detecting the carrier state of females in the family is challenging due to the presence of the normal allele. Different methods like linkage analysis, real time PCR and MLPA have been used to assess the carrier status in this situation. Linkage analysis is limited by the availability of informative markers and adequate number of family members. Real time PCR involves standardisation and preparation of calibration curves for each run. Although MLPA is a better alternative, it can be time consuming and involves multiple steps. We have therefore developed a fluorescent PCR based gene dosage approach which is simple, rapid and cost-effective for determining the carrier status of females in families with deletions in the F9 gene. 200ng of DNA extracted by standard protocols was used for amplification with primers designed to amplify a 160bp product from exon h in the F9 gene. One of the primers was fluorescently labelled. Amplification was carried out using these primers for 20 cycles only and the amplified product was subjected to capillary electrophoresis on an ABI 310 genetic analyser. A 230 bp fragment in the albumin gene was used as the control. Analysis was done using Genescan and Genotyper software. The ratio between the peak heights of the exon h in the F9 and control genes in the patient samples were normalised to a normal control. Five families with deletional HB were analysed (in toto deletion-1; Ex g-h – 1; Ex g-poly A-1; Ex h-poly A-2). The ratios in the probands and the family members are presented in the table. Out of eight females analysed, 6 were carriers and 2 were normal. The mean ratio in the carriers was 0.49±0.08 and 0.75±0.05 in the normal. Deletions are not uncommon in HB and deletions involving the exon g and h constitute a major group. Among 212 families with HB assessed at our centre, we have identified large deletions in 8 families (3.7%). It is interesting to note that all except one of these deletional mutations involved exon h. This method confirmed the presence of these deletions in the males and helped us to identify the carrier status of the females in the family. Identification of carrier status of females with deletions in F9 gene by gene dosage Subject ID Peak height of Exh in F9 Peak height of albumin Normalised Ratio Interpretation HB5 284 442 0.59 Carrier HB6 305 489 0.57 Carrier HB22 188 372 0.47 Carrier HB63 85 165 0.48 Carrier HB129 247 295 0.78 Normal HB238 94 326 0.4 Carrier HB280 372 679 0.77 Normal HB384 202 670 0.4 Carrier Disclosures: No relevant conflicts of interest to declare.

scholarly journals Screening of Dystrophin Gene Deletions in Egyptian Patients with DMD/BMD Muscular Dystrophies

2000 ◽  
Vol 16 (3-4) ◽  
pp. 125-129 ◽  
Author(s):  
Laila K. Effat ◽  
Ashraf A. El-Harouni ◽  
Khalda S. Amr ◽  
Tarik I. El-Minisi ◽  
Nagwa Abdel Meguid ◽  
...  
Keyword(s):  
Muscular Dystrophy ◽  
Dna Analysis ◽  
Human Genetics ◽  
Hot Spot ◽  
Cost Effective ◽  
Dystrophin Gene ◽  
Becker Muscular Dystrophy ◽  
Pedigree Analysis ◽  
Gene Deletions ◽  
Serum Creatine Phosphokinase

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations within the dystrophin gene. Our study has identified 100 Egyptian families collected from the Human Genetics Clinic, National Research Center, Cairo. All cases were subjected to complete clinical evaluation pedigree analysis, electromyography studies, estimation of serum creatine phosphokinase enzyme (CPK) levels and DNA analysis. Multiplex PCR using 18 pairs of specific primers were used for screening of deletion mutations within the dystrophin gene. A frequency of 55% among the families. Sixty per cent of detected deletions involved multiple exons spanning the major or the minor hot spot of the dystrophin gene. The remainder 40% which mainly involved exon 45. Comparing these findings with frequencies of other countries it was found that our figures fall within the reported range of 40%– for deletions. The distribution of deletions in our study and other different studies was variable and specific ethnic differences do not apparently account for specific deletions. In addition this study concluded that employment of the 18 exon analysis is a cost effective and a highly accurate (97% to launch a nationwide program.

scholarly journals An Optimized Real-Time qPCR Method for the Effective Detection of Human Malaria Infections

Diagnostics ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 736
Author(s):  
Saiful Arefeen Sazed ◽  
Mohammad Golam Kibria ◽  
Mohammad Shafiul Alam
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Plasmodium Species ◽  
Cost Effective ◽  
Plasmodium Malariae ◽  
Cross Reactivity ◽  
Diagnostic Pcr ◽  
Pcr Method ◽  
Human Plasmodium

Polymerase chain reaction, although an expensive method for the detection of human Plasmodium spp., is still considered the finest for the diagnosis of malaria. The conventional diagnostic PCR is an inexpensive process but consumes a lot of time, reagents and lacks sensitivity. On the other hand, real-time PCR assays currently being used are mostly probe-based expensive methods and sometimes not feasible to detect all the species in a single amplification reaction condition. Here we have established a real-time PCR method that is time and cost effective with a single protocol to detect and distinguish five human Plasmodium species using the existing primers efficiently. The primers used here are being used in the conventional method and the sensitivity as well as specificity of this method has also been immensely improved (100%). The lower limit of detection for Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae are 0.064 parasites/µL, 1.6 parasites/µL, and 0.32 parasites/µL respectively and no cross reactivity was observed. Besides, we have analyzed melt curves that can be used for further species confirmation and validation purposes using multiplex systems. This method, therefore, can be considered as an alternative to the existing lineup for molecular diagnosis of malaria in endemic countries.

Determination of Cry1Ac copy number in transgenic pigeonpeaplants using quantitative real time PCR.

2016 ◽  
Author(s):  
Meenakshi . Jain ◽  
Surender . Khatodia ◽  
Pushpa . Kharb ◽  
Praveen . Batra ◽  
Vijay K. Chowdhury
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Cost Effective ◽  
Single Copy ◽  
Quantitative Real Time Pcr ◽  
Real Time Quantitative Pcr ◽  
Specificity And Sensitivity ◽  
Transgenic Lines ◽  
Pcr Method

Copy number of Cry1Ac in transgenic pigeonpea plants was determined by quantitative real time PCR using Syber Green as fluorescence indicator. Gene specific primers designed to amplify relatively long amplicons (400 – 600 bp), for Cry1Ac was used to increase specificity and sensitivity of Real time PCR. Estimated copy number in transgenic lines using real-time quantitative PCR and southern hybridization was correlated and found to be same i.e. single copy number. This study shows effectivness of real-time PCR method for estimating the transgene copy number in transgenic pigeonpea plants by a simple, accurate and cost effective manner.Keywords: Copy number, Cry1Ac, Pigeonpea transformation, Real-time PCR, Syber green.

Detection of Streptococcus pneumoniae in Sputum Samples by Real- Time PCR.

2020 ◽  
Vol 18 ◽  
Author(s):  
Pegah Shakib ◽  
Mohammad Reza Zolfaghari
Keyword(s):  
Streptococcus Pneumoniae ◽  
Real Time ◽  
Real Time Pcr ◽  
False Negative ◽  
Cross Sectional Study ◽  
Laboratory Culture ◽  
Cross Sectional ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Method

Background: Conventional laboratory culture-based methods for diagnosis of Streptococcus pneumoniae are time-consuming and yield false negative results. Molecular methods including real-time (RT)-PCR rapid methods and conventional PCR due to higher sensitivity and accuracy have been replaced instead traditional culture assay. The aim of the current study was to evaluate lytA gene for detection of Streptococcus pneumoniae in the cerebrospinal fluid of human patients with meningitis using real-time PCR assay. Material and Methods: In this cross-sectional study, a total of 30 clinical specimens were collected from patients in a period from September to December 2018. In order to evaluate the presence of lytA gene, conventional and real-time PCR methods were used without culture. Results: From 30 sputum samples five (16.66%) isolates were identified as S. pneumoniae by lytA PCR and sequencing. Discussion: In this research, an accurate and rapid real-time PCR method was used, which is based on lytA gene for diagnosis of bacteria so that it can be diagnosed. Based on the sequencing results, the sensitivity for detection of lytA gene was 100% (5/5).

scholarly journals Real-Time PCR as an Alternative Technique for Detection of Dermatophytes in Cattle Herds

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1662
Author(s):  
Dominik Łagowski ◽  
Sebastian Gnat ◽  
Aneta Nowakiewicz ◽  
Aleksandra Trościańczyk
Keyword(s):  
Light Microscopy ◽  
Real Time ◽  
Real Time Pcr ◽  
Direct Analysis ◽  
Carrier Status ◽  
The Real ◽  
Detection Techniques ◽  
Direct Light ◽  
Microscopy Analysis ◽  
Cattle Herds

Dermatophytes are filamentous fungi with the ability to digest and grow on keratinized substrates. The ongoing improvements in fungal detection techniques give new scope for clinical implementations in laboratories and veterinary clinics, including the monitoring of the disease and carrier status. The technologically advanced methods for dermatophyte detection include molecular methods based on PCR. In this context, the aim of this study was to carry out tests on the occurrence of dermatophytes in cattle herds using qPCR methods and a comparative analysis with conventional methods. Each sample collected from ringworm cases and from asymptomatic cattle was divided into three parts and subjected to the real-time PCR technique, direct light microscopy analysis, and culture-based methods. The use of the real-time PCR technique with pan-dermatophyte primers detected the presence of dermatophytes in the sample with a 10.84% (45% vs. 34.17%) higher efficiency than direct analysis with light microscopy. Moreover, a dermatophyte culture was obtained from all samples with a positive qPCR result. In conclusion, it seems that this method can be used with success to detect dermatophytes and monitor cowsheds in ringworm cases and carriers in cattle.

scholarly journals Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

2003 ◽  
Vol 69 (12) ◽  
pp. 7430-7434 ◽  
Author(s):  
Trevor G. Phister ◽  
David A. Mills
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Dekkera Bruxellensis ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Spoilage Yeast ◽  
Pcr Method ◽  
26S Rrna

ABSTRACT Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis.

Development and application of a novel reverse transcription real-time PCR method for miR-499 quantification

2013 ◽  
Vol 46 (15) ◽  
pp. 1566-1571 ◽  
Author(s):  
Weidong Zheng ◽  
Yuwei Di ◽  
Yinghong Liu ◽  
Ge Huang ◽  
Youwei Zheng ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
Pcr Method

MGB-based one-step multiplex real-time PCR method for rapid detection of HPV

2013 ◽  
Vol 12 (3) ◽  
pp. 107-113 ◽  
Author(s):  
Jie Huang ◽  
Chun Gao ◽  
Xilai Ding ◽  
Shoufang Qu ◽  
Licheng Liu ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
One Step ◽  
Pcr Method

scholarly journals Rapid concentration and sensitive detection of hookworm ova from wastewater matrices using a real-time PCR method

2015 ◽  
Vol 159 ◽  
pp. 5-12 ◽  
Author(s):  
P. Gyawali ◽  
J.P.S. Sidhu ◽  
W. Ahmed ◽  
P. Jagals ◽  
S. Toze
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Sensitive Detection ◽  
Pcr Method ◽  
Hookworm Ova
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