Real-time quantitative PCR detection of Escherichia coli O157:H7

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1 Real-time quantitative PCR detection of Escherichia coli O157:H7 Chen Si y, Huang Kun-Lun y, Xu Wen-Tao, Li Yuan and Luo Yun-Bo* College of Food Science and Nutritional Engineering, China Agricultural University, Beijing , China Received 29 April 2005; Accepted 23 May 2005 First published in Journal of Agricultural Biotechnology 2006, 14(5): Abstract A rapid and accurate real-time quantitative polymerase chain reaction (real-time PCR) method with SYBR Green I was established for detecting Escherichia coli O157:H7. A pair of primers were designed to amplify the eae gene. The dissociation curves showed that the amplification product was very specific. The optimal conditions and standard curve were established. The result indicated that real-time PCR was 1000 times more sensitive than ordinary PCR. Keywords: Escherichia coli O157:H7; SYBR Green I; quantitative fluorescence; real-time PCR Introduction Escherichia coli O157:H7 is the most common type of enterohaemorrhagic E. coli (EHEC). During the 20 years since the first isolation of E. coli O157:H7 (Riley et al., 1983), many reports of O157:H7 outbreaks and spread have been published in Canada, the UK, Japan, etc. The largest outbreak in the world took place in Japan, where more than 9000 persons were infected and more than 10 died (Infectious Agents Surveillance Center, 1996). The first case of E. coli O157:H7 in China was detected in Jiangsu province in 1987 (Wang and Shi, 2001). Later, this infection was reported in many other places, for example Henan province (Zhang et al., 2003a), Shanghai (Gu et al., 2003) and Hebei province (Zhang et al., 2003b). The infective dose of O157:H7 is extremely low. Some reports show that fewer than 10 bacteria would cause disease (Paton and Panton, 1998). There is no specifically effective vaccine at present, so research on its prevention and incidence is vital. Methods of O157 detection are mainly traditional isolation and incubation, immunology and polymerase chain reaction (PCR) at the molecular level. An advanced comparative method is real-time fluorescence quantitative PCR, which involves the use of a fluorescence probe or dye to reveal the quantities of template DNA in real time, and to display this graphically. Results recorded with this technique are often accurate and Bhagwat (2003) used it to increase the lowest limit of detection of EHEC O157:H7 to 1 10 cells/ml. By combining real-time PCR and molecular labelling technology, Fortin (2001) detected 1 cfu (colony forming unit)/ml. However, no similar reports have been published in Chinese laboratories. In this paper, the eaea gene from Escherichia coli O157:H7 cultured on intestinal epidermal cells was amplified to establish a method for detecting O157:H7 by fluorescence quantitative PCR. Materials and methods Materials Bacteria E. coli O157:H7 EDL933 (American) was donated by Jing Huai-qi (Epidemic Disease Microbiology Research Institute of China Preventive Medical Science Academy). Negative control E. coli BL21 and DH5a were from the Food Biotechnology Laboratory of Food Science College (China Agricultural University, China).

2 Instruments The following equipment was utilized: ABI Prism (r) 7000 fluorescence quantitative PCR (Applied Biosystems Co., Foster City, California, USA); U-Vikon XL UV/VIS spectrophotometer (Secomam Co., France); PCR analyser (Hybaid Ltd, Britain) Main reagents SYBR Green I PCR kit was purchased from Beijing Tian Wei Shi Dai Science Technology Co. (Beijing, China); Taq DNA polymerase from Beijing Ding Guo Biotechnology Co. (Beijing, China). The primers were synthesized by Shanghai Sheng Gong Biotechnology Co. (Shanghai, China). Primer order: Forward, K14924: 5 0 -TTACCAGCGATACCAAGAGC-3 0 Reverse, K14925: 5 0 -CAACATGACCGATGACAAGG-3 0 The length of PCR product was 126. Methods Preparation of template DNA Single colonies from bacterial cultures were incubated at 37 C on a shaker for 10 h. After bacterial density counting by electronic microscopy, the DNA was extracted with phenol chloroform according to Li (1994). DNA quality was identified using UV/VIS spectrophotometry, diluted 10 times, and cfu/ml concentration gradients were used as templates. Qualitative PCR amplification The reaction system contained 25 ml: 1rbuffer (10 mmol/ l Tris HCl, ph 8.3, 50 mmol/l KCl, 2 mmol/l MgCl 2 ), 0.2 mmol/l deoxynucleoside triphosphates (dntps), 1 U Taq DNA polymerase, 2 mmol/l forward and reverse primers and 2r10 6 cfu/ml as DNA template. The PCR program initially started with denaturation at 95 C for 10 min, followed by 40 cycles of 94 C for 30 s, 59 C for 45 s, 72 C for 30 s, and 72 C final extension for 10 min. Negative bacterium and water control samples were set up to examine the specificity, and the reaction sensitivity was measured by the 10 concentration gradient templates. The amplified products were examined by 2% agar gel electrophoresis. Fluorescence quantitative PCR amplification The reaction system (40 ml) comprised 20 ml SYBR Green I mixture (2 ml 10rbuffer, 0.1 mol/l dntps, 2.5 U Taq DNA polymerase), 2 mmol/l forward and reverse primers and DNA template. The PCR program initially started with a 95 C denaturation for 10 min, followed by 40 cycles of 94 C for 15 s, 60 C for 30 s, 72 C for 30 s. The fluorescence was measured during the annealing period of each cycle. After reaction completion, the product was heated at 95 C, cooled down to 60 C, then slowly reheated to 95 C (0.2 C/s). The changes in fluorescence signal and the dissociation curve of the amplified product were recorded. Specificity examination and reaction sensitivity measurement were conducted as above. Optimization of the fluorescence quantitative PCR reaction To determine the optimal primer concentration, 0.5, 1.0, 2.0, 3.0 and 5.0 ml primer were added and amplified, respectively. Different DNA concentrations were used to determine the optimal range of quantitative PCR template. Preparation of a fluorescence quantitative standard curve Optimal DNA template concentration was determined based on the influence of different template concentrations on amplification and fluorescence. Reaction systems of five template gradients in the optimal concentration range were as described above under Fluorescence quantitative PCR amplification. After the threshold and baseline were determined, a standard curve was obtained. Results 126 Qualitative PCR reaction Fig. 1. Qualitative polymerase chain reaction (PCR) for the eae gene. Lanes: 1, E. coli strain EDL933; 2, E. coli strain BL21; 3, control (water); 4, DL2000 marker. The ratio of OD 260 /OD 280 by UV spectrophotometry was 1.8, indicating the purity of the DNA template. Qualitative PCR for the eae gene (Fig. 1) showed that only the positive bacteria EDL933 exhibited a band of 126. No

1 2 3 4 5 6 7 8 126 amplification was observed in the negative bacteria BL21 or the water control sample, which showed that the designed primers reached the PCR detection requirements and no

The first band (10 3 cfu/ml) was the lowest template concentration detected by normal PCR.

3), and reached the requirements of quantitative detection, with a comparatively high peak and long index increase period.

3 amplification was observed in the negative bacteria BL21 or the water control sample, which showed that the designed primers reached the PCR detection requirements and no operational mistake occurred during the procedure. As indicated in Fig. 2, the intensity of the bands decreased with the decrease of template concentration. The first band (10 3 cfu/ml) was the lowest template concentration detected by normal PCR. Fluorescence quantitative PCR amplification The fluorescence signal curve of positive bacteria EDL933 was smooth and stable (Fig. 3), and reached the requirements of quantitative detection, with a comparatively high peak and long index increase period. No amplification was noticed from samples of corresponding negative bacteria (BL21 and DH5a) and water control, indicating good specificity of the designed primer in realtime PCR reaction. This was confirmed by the profile obtained using agar gel electrophoresis (result not shown), which was similar to that of Fig. 1. Quantitative dissociation curves of 10 DNA template concentrations ( cfu/ml) were performed using realtime fluorescence. It can be seen from the curves (Fig. 4) that all the concentration peaks were significantly pure and without primer dimers, proving once more the product specificity. The 1 cfu/ml concentration curve indicated that the detection limit, by real-time PCR, could theoretically reach 1 cfu/ml, which is 1000 times higher than in the normal quantitative PCR. Optimization of fluorescence quantitative PCR reaction Fig. 2. Sensitivity analysis of ordinary polymerase chain reaction (PCR). Lanes 1 7, cfu/ml E. coli strain EDL933, respectively; 8, DL2000 marker. A concentration of primers that is too high may cause primer dimers, and one that is too low will influence the amplification efficiency. The final determined primer Relative fluorescence absorption concentration here was 2 mmol/l for both forward and reverse primers. An excessively high or low concentration of template can influence the peak of the dissociation curves and the S-shaped curve of fluorescence absorption. In this trial, the optimal concentration for detecting E. coli O157:H7 ranged between 10 2 and 10 7 cfu/ml, with a sample volume of 2 ml. Standard curve Cycle Number Fig. 3. Specificity analysis for real-time PCR. EDL933, positive bacteria; BL21 and DH5a, negative bacteria; ck, water control. (See online for a colour version of this Fluorescence signal intensity Temperature ( C) Fig. 4. Quantitative dissociation curves of 10 DNA template concentrations ( cfu/ml). CK, water control. (See online for a colour version of this According to the above result, five DNA template concentrations (10 2,10 3,10 4,10 5 and 10 6 cfu/ml) were selected for amplification (Fig. 5) and the standard curve (Fig. 6) was produced, based on the threshold, baseline and cycle threshold (Ct) values. The horizontal axis in Fig. 6 is the log value of the concentration of positive bacteria EDL933, and the vertical axis is the Ct value detected by real-time PCR.

4 Relative fluorescence absorption Cycle Number products as well as the existence, or not, of dimers. However, this technique can not be used for multiple detection and the fluorescence signal does not show a template specificity. The utilization of this technique in this research has lowered the experimental cost and has allowed the application of dissociation curves to the analysis, and the unique primer design minimized any disadvantages. Determination of the quantitative unit Fig. 5. Fluorescence curves of cfu/ml of E. coli strain EDL933. (See online for a colour version of this Ct Since the present study concerns the toxin-producing E. coli O157:H7, the total colony amount is much more important than its total DNA. Here, the absolute quantity of DNA was not adopted as the quantitative unit; the choice of cfu as the quantitative unit best reflects bacterial vigour and is more meaningful in microbe detection. Selection of standard substances The equation of the final standard curve was y =-2.367x The quantity of the positive bacteria in samples could be calculated by inserting detected Ct values (y) into the equation. The relative coefficient R 2 was over 0.98, indicating that this equation can be used as the standard equation for O157 detection in the future. Discussion Y = X Log CO Fig. 6. Standard curve Ct, cycle threshold; CO, concentration of bacteria. (See online for a colour version of this Advantages and disadvantages of SYBR Green I fluorescence dye During a dye detection procedure, a certain number of dye molecules can be combined with a DNA doublechain as it forms. A fluorescence signal will be enhanced as the signal intensity and DNA quantity are directly proportional. The dye method is advantageous as it presents a low-cost method, with no need of a probe and being suitable for primary detection. Furthermore, dissociation curves can quantitatively determine the PCR The basic principles in choosing standard substances are: PCR efficiency of substance similar to that of the sample the less the difference the less the error; use under the same conditions as the sample (same instruments, reagents, recycling index, and experiment time); use the same quality templates and the same T m (melting temperature) primer. In this paper, the standard curve was determined by fluorescence quantitative amplification of diluted positive bacteria, which guarantees the identity of standard substances and samples. Threshold and baseline function A good choice of the threshold and baseline can help in drawing the standard curves. By adjusting the values of these two indexes, the relative coefficient of the standard curve reaches more than Acknowledgements This research was supported by Key Technology of Food Safety of The Tenth Five-Year Plan, Ministry of Science and Technology, China (No. 2001BA804A23-1). References Bhagwat AA (2003) Simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella strains by real-time PCR. International Journal of Food Microbiology 84(2):

5 Fortin NY (2001) Use of real-time polymerase chain reaction and molecular beacon for the detection of Escherichia coli O157:H7. Analytical Biochemistry 289(2): Gu BK, Xu XB, Jin HM, Hu PY and Xi MF (2003) Surveillance of E. coli O157:H7 in meat food of Shanghai district. Disease Surveillance 8(1): 5 7 (in Chinese with English abstract). Infectious Agents Surveillance Center, National Institute of Health and Infectious Diseases Control Division, Ministry of Health and Welfare, Japan (1996) Outbreaks of enterohemorrhagic Escherichia coli O157:H7 infection. Japan ISAR 17(8): Li DB (1994) Principle and Methods of Recombinant DNA. Zhejiang: Science and Technology Press, pp (in Chinese). Paton AW and Panton JC (1998) Detection and characterization of shiga toxin genic Escherichia coli by using multiplex PCR assays for stx1 stx2 enterohemorrhagic E. coli hly A, rfbo 111 and rfbo157. Journal of Clinical Microbiology 36(2): Riley LW, Remis R, Helgerson SD, et al. (1983) Outbreaks of hemorrhagic colitis associated with a rare Escherichia coli serotype. New England Journal of Medicine 308: Wang H and Shi ZY (2001) Study on epidemiology of E. coli O157:H7. Jiangsu Health Care 3(1): 4 5 (in Chinese with English abstract). Zhang J, Xia SL and Ma H (2003a) The epidemiological investigation on infectious cases of shiga s toxin-producing E. coli O157:H7 in eastern Henan, China. Strait Journal of Preventive Medicine 9(5): (in Chinese with English abstract). Zhang YL, Guo YX, Li GY, et al. (2003b) Surveillance of E. coli O157:H7 infection in Hebei province. Disease Surveillance 8(3): (in Chinese with English abstract).

Pr oject Summar y. Rapid quantification of culturable and viable-but-nonculturable Escherichia coli O157:H7 in beef products using EMA-Real Time PCR

Pr oject Summar y. Rapid quantification of culturable and viable-but-nonculturable Escherichia coli O157:H7 in beef products using EMA-Real Time PCR Pr oject Summar y Rapid quantification of culturable and viable-but-nonculturable Escherichia coli O17:H7 in beef products using EMA-Real Time PCR Principal Investigator: Azlin Mustapha University of Missouri