1. Result Introduction Methods
Standard curve quantification for bacterial DNA using a real-time PCR assay
Jannatul Ferdous1,2 , Zenat Zebin Hossain1,2, Suhella Tulsiani2,3, Peter Kjær Mackie Jensen2,3, Anowara Begum1*
1 Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh 2Department of International Health, Immunology and Microbiology, University of Copenhagen, Denmark Copenhagen Centre for Disaster Research, University of Copenhagen, Denmark 1871 3
Result 1
Introduction
Amplification plot from linear graph type was obtained for both standards and unknown spiked samples. Ct values and baselines of each amplification curve for all standard samples met the dilution series of DNA (Figure-2). Quantification of unknown spiked samples were achieved from the standard curve which was quantified by Step One machine software.
PCR efficiency
PCR efficiency for uidA gene and ctxA gene were obtained % and 98.08% respectively. In this experiment, high-efficiency condition results in better sensitivity at low target concentrations. This method enables to detect as low as 3 copy number per reaction.
Real-time PCR or quantitative PCR (qPCR) is a dynamic and cogent assay for the detection and quantification of specified nucleic acid sequences compared to both, traditional culture techniques and ‘end point’ conventional PCR for environmental samples
Knowledge gap
Limitations of quantification by cell culture dilution are
(i) Loss of viable but non cultural cells which gives varying number of cfu count that shows variation with the copy number present in the samples
(ii) Concentration of extracted DNA from serial dilution of cells is not equivalent to cfu count found from serial dilution of cells
Goal of the study
To develop a method for real time quantification of exact copy no of bacteria in a sample based on serial dilution of DNA.
3.04E6
3.04E5
3.04E4
3.04E3
304
30.4
3.04 2
Methods
Figure 1: Study design of qPCR by DNA dilution method
In this method, to generate a standard curve in qPCR, total bacterial DNA was diluted in a 10-fold series and each sample was correlated with its estimated cell count. To accurately determine the efficiency of this PCR method two replicates of standards and 6-log dilutions were performed.
Different autoclaved water samples spiked separately with 10 µl of pure culture of E. coli and V. cholerae were used as unknown samples to observe the bacterial cell count from this standard curve experiment.
Figure 2: Amplification plot (∆Rn vs Cycle) of ctxA gene for V. cholerae with estimated copy number for each dilution. Blue bar shows the threshold value(Ct) .
Figure 4: Plate layout of qPCR showing hypothetically inputted copy no of V. cholerae per standard curve well and the calculated copy no. in unknown well (highlighted in red). S= Standard, U= Unknown.
The detection and quantification of pathogens are important when assessing the health risks associated with food and water. This method allowed us to quantify accurately the levels of fecal contamination indicators, such as Escherichia coli and the VBNC cells of Vibrio cholerae which is one of the leading causes of life threatening diarrheal disease in Bangladesh.
Determination of cell no. from DNA mass:
For Escherichia coli, measured concentration of starting bacterial DNA = ng/µl, since, 5.16 fg DNA= one Escherichia coli genome mass = one Escherichia coli cell. So, ng/µl = 1.86X107 cells of E. coli
For Vibrio cholerae, measured concentration of starting bacterial DNA = ng/µl, since, 4.52 fg DNA= one Vibrio cholerae genome mass = one Vibrio cholerae cell. So, ng/µl = 1.74X107 cells of Vibrio cholerae
Figure 3: Standard curves using ctxA qPCR showing correlation between the log value of 10-fold serial dilutions of V. cholerae DNA and respective Ct values. The red dots represent the standard samples and the blue dot represents the unknown sample.