Detection and Real-time PCR Assay for the Quantification of Carbapenemase Gene blablaNDM-1 in Hospital Effluent

This study aims to isolate gram-negative bacteria (GNB) harboring the gene NDM-1 from the tertiary care hospital effluents. Also, aims to evaluate the relative copy number of blaNDM-1 carried by the positive isolates. The study isolated 215 GNB from 40 effluent samples. The antibiotic susceptibility tests for carbapenems were performed using disc diffusion assay. The isolates resistant to either meropenem or imipenem were checked for the existence of MBL by phenotypic methods. The isolates carrying NDM-1 gene were genotypically confirmed by Polymerase chain reaction (PCR). The gene copy number of blaNDM- were determined by quantative real-time PCR. A total of 22 isolates showed phenotypic resistance to carbapenems and were characterized by biochemical methods. Among them, 12 harbored NDM-1 gene by PCR; these bacteria were subjected to qPCR for determining the absolute copy numbers of the NDM-1 gene on it. The gene abundance in the strains was in the range of 3.28× 105 to 6.05× 106 copies/ ng of DNA. Hospital effluents are important pool of antibiotic-resistant bacteria harboring the blaNDM-1 and infections caused by these bacteria are difficult to treat. Hence, the present study stresses the need for stringent antibiotic use and efficient wastewater treatment policies in these hospital settings, which is paramount in achieving sustainable health.

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Diagnosis of haploidy and triploidy based on measurement of gene copy number by real-time PCR

Human Mutation ◽

10.1002/1098-1004(200011)16:5<431::aid-humu8>3.0.co;2-z ◽

2000 ◽

Vol 16 (5) ◽

pp. 431-436 ◽

Cited By ~ 49

Author(s):

Klaus Wilke ◽

B�lent Duman ◽

J�rgen Horst

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Copy Number ◽

Gene Copy Number ◽

Gene Copy

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TaqMan real-time PCR quantification strategy of CYP2D6 gene copy number for the LightCycler 2.0

Clinica Chimica Acta ◽

10.1016/j.cca.2009.03.007 ◽

2009 ◽

Vol 403 (1-2) ◽

pp. 207-211 ◽

Cited By ~ 14

Author(s):

Duc L. Nguyen ◽

Julia Staeker ◽

Barbara Laika ◽

Werner Steimer

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Copy Number ◽

Gene Copy Number ◽

Gene Copy ◽

Cyp2d6 Gene

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Photo-Induced Electron Transfer Real-Time PCR for Detection ofPlasmodium falciparum plasmepsin 2Gene Copy Number

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00317-18 ◽

2018 ◽

Vol 62 (8) ◽

Cited By ~ 1

Author(s):

Samaly Santos Souza ◽

Mariangela L'Episcopia ◽

Carlo Severini ◽

Venkatachalam Udhayakumar ◽

Naomi W. Lucchi

Keyword(s):

Electron Transfer ◽

Real Time ◽

Real Time Pcr ◽

Copy Number ◽

Gene Copy ◽

Content Type ◽

Copy Numbers ◽

Ofplasmodium Falciparum ◽

Photo Induced Electron Transfer ◽

Gene Copy Numbers

ABSTRACTPiperaquine is an important partner drug used in artemisinin-based combination therapies (ACTs). An increase in theplasmepsin 2and3gene copy numbers has been associated with decreased susceptibility ofPlasmodium falciparumto piperaquine in Cambodia. Here, we developed a photo-induced electron transfer real-time PCR (PET-PCR) assay to quantify the copy number of theP. falciparumplasmepsin 2gene (PfPM2) that can be used in countries whereP. falciparumis endemic to enhance molecular surveillance.

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Sensitive and specific real-time PCR assays to accurately determine gene copy-number variations (CNVs) of human complement C4A, C4B, C4-Long, C4-Short and RCCX modules: Elucidation of C4 GCNVs in 50 consanguineous subjects with defined HLA genotypes

Molecular Immunology ◽

10.1016/j.molimm.2007.06.034 ◽

2007 ◽

Vol 44 (16) ◽

pp. 3921

Author(s):

Yee-Ling Wu ◽

Stephanie L. Savelli ◽

Yan Yang ◽

Bi Zhou ◽

Brad H. Rovin ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Copy Number ◽

Gene Copy Number ◽

Copy Number Variations ◽

Gene Copy ◽

Human Complement ◽

Hla Genotypes ◽

Pcr Assays

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27P Quantitative Analysis by Real-Time Pcr for ESR1 Gene Copy Number in Serum in Breast Cancer Samples

Annals of Oncology ◽

10.1016/s0923-7534(19)65673-5 ◽

2012 ◽

Vol 23 ◽

pp. ii22

Author(s):

J. Martinez-Galan ◽

B. Torres-Torres ◽

J.R. Delgado ◽

M.I. Núñez ◽

S. Ríos

Keyword(s):

Breast Cancer ◽

Quantitative Analysis ◽

Real Time ◽

Real Time Pcr ◽

Copy Number ◽

Gene Copy Number ◽

Gene Copy ◽

Esr1 Gene

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A Simple Multiplex Real-Time PCR Methodology for the SMN1 Gene Copy Number Quantification

Genetic Testing and Molecular Biomarkers ◽

10.1089/gtmb.2008.0084 ◽

2009 ◽

Vol 13 (1) ◽

pp. 37-42 ◽

Cited By ~ 11

Author(s):

Nadia Passon ◽

Federico Pozzo ◽

Cristiano Molinis ◽

Elisa Bregant ◽

Cinzia Gellera ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Copy Number ◽

Gene Copy Number ◽

Gene Copy ◽

Smn1 Gene

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Identification and copy number profiling of sex chromosome specific gene fragments in crossbred pigs with numerically normal karyotype

Animal Production Science ◽

10.1071/an19287 ◽

2020 ◽

Vol 60 (9) ◽

pp. 1145

Author(s):

Vandana Yadav ◽

Nihar Ranjan Sahoo ◽

Pushpendra Kumar ◽

G. K. Gaur ◽

A. P. Sahoo ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Copy Number ◽

Sex Chromosome ◽

Normal Karyotype ◽

Autosomal Gene ◽

Metastasis Suppressor ◽

Quantitative Real Time Pcr ◽

Relative Copy Number ◽

Crossbred Pigs

We examined the copy number profile of sex chromosome specific genes in crossbred pigs with numerically normal karyotype. A total of 30 (15M + 15F) Landlly (Landrace crossbred) pigs from a research farm were cyto-screened by karyotyping to determine the number of chromosomes per diploid cell using short-term peripheral lymphocyte culture technique. All pigs had numerically normal karyotype with 38, XX (female) and 38, XY (male). Genomic DNA was extracted from cyto-screened pigs of both sexes. A set of sex chromosome (X and Y) specific single copy gene fragments along with an autosomal gene fragment were selected out of 12 pairs of primers on the basis of male specific PCR amplification, PCR and qPCR specificity. Quantitative real-time PCR was performed to study the relative copy number change of selected Phosphate repressible alkaline phosphatase X linked (PHOX) gene, using the Eukaryotic Translation Initiation Factor 1A Y-Linked (EIF1AY) gene as control and Breast Cancer Metastasis-Suppressor 1-Like (BRMS1 L- autosomal gene) as the reference gene. The relative copy number of the PHOX gene in females was found to be 1.873 times higher compared with the EIF1AY gene in males. The present study indicates that the real-time quantitative real-time PCR based copy number analysis can be helpful for the detection of sex chromosome ratio in pigs to aid as a preliminary screening for numerical sex chromosomal aneuploidies, adding to the rate of throughput of traditional cyto-screening.

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An investigation into gene copy number determination in transgenic yeast; The importance of selecting a reliable real-time PCR standard

Biologicals ◽

10.1016/j.biologicals.2020.04.001 ◽

2020 ◽

Vol 65 ◽

pp. 10-17

Author(s):

Roghayeh Shirvani ◽

Mohammad Barshan-tashnizi ◽

Maryam Shahali

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Copy Number ◽

Gene Copy Number ◽

Gene Copy ◽

Copy Number Determination ◽

Transgenic Yeast

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Real-time PCR for the detection of precise transgene copy number in durum wheat

Cellular & Molecular Biology Letters ◽

10.2478/s11658-011-0029-5 ◽

2011 ◽

Vol 16 (4) ◽

Cited By ~ 18

Author(s):

Agata Gadaleta ◽

Angelica Giancaspro ◽

Maria Cardone ◽

Antonio Blanco

Keyword(s):

Durum Wheat ◽

Real Time ◽

Real Time Pcr ◽

Copy Number ◽

Gene Copy Number ◽

Correlation Coefficients ◽

Gene Copy ◽

Transgene Copy Number ◽

Locus Number ◽

Wheat Lines

AbstractRecent results obtained in various crops indicate that real-time PCR could be a powerful tool for the detection and characterization of transgene locus structures. The determination of transgenic locus number through real-time PCR overcomes the problems linked to phenotypic segregation analysis (i.e. lack of detectable expression even when the transgenes are present) and can analyse hundreds of samples in a day, making it an efficient method for estimating gene copy number. Despite these advantages, many authors speak of “estimating” copy number by real-time PCR, and this is because the detection of a precise number of transgene depends on how well real-time PCR performs.This study was conducted to determine transgene copy number in transgenic wheat lines and to investigate potential variability in sensitivity and resolution of real-time chemistry by TaqMan probes. We have applied real-time PCR to a set of four transgenic durum wheat lines previously obtained. A total of 24 experiments (three experiments for two genes in each transgenic line) were conducted and standard curves were obtained from serial dilutions of the plasmids containing the genes of interest. The correlation coefficients ranged from 0.95 to 0.97. By using TaqMan quantitative real-time PCR we were able to detect 1 to 41 copies of transgenes per haploid genome in the DNA of homozygous T4 transformants. Although a slight variability was observed among PCR experiments, in our study we found real-time PCR to be a fast, sensitive and reliable method for the detection of transgene copy number in durum wheat, and a useful adjunct to Southern blot and FISH analyses to detect the presence of transgenic DNA in plant material.

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