Tomohiko Ikeuchi1, Masafumi Seki2,3*, Yukihiro Akeda4, Norihisa Yamamoto2, Shigeto Hamaguchi2, Tomoya Hirose5, Keiichiro Yamanaka1, Masato Saito1, Kazunori Tomono2 and Eiichi Tamiya1
1Department of Applied Physics, Graduate School of Engineering, Japan
2Division of Infection Control and Prevention, Osaka University Hospital, Japan
3Division of Respiratory Medicine and Infection Control, Tohoku Pharmaceutical University Hospital, Sendai, Japan
4Laboratory of Clinical Research on Infectious Diseases, Research Institute for Microbial Diseases, Japan
5Department of Traumatology and Acute Critical Medicine, Osaka University Hospital, Osaka University, Suita City, Osaka, Japan
Received: 24 September, 2015; Accepted: 18 November, 2015; Published: 20 November, 2015
Masafumi Seki, MD, PhD, Division of Respiratory Medicine and Infection Control, Tohoku Pharmaceutical University Hospital, 1-12-1 Fukumuro, Miyagino, Sendai, 983-8512, Japan, Tel: +81-22-259-1221; Fax: +81-22-259-0507; E-mail:
Ikeuchi T, Seki M, Akeda Y, Yamamoto N, Hamaguchi S, et al.(2016) PCR-Based Method for Rapid and Minimized Electrochemical Detection of mecA Gene of Methicillin-Resistant Staphylococcus aureus and Methicillin-Resistant Staphylococcus epidermis. Glob J Infect Dis Clin Res 2(1): 008-012. DOI: 10.17352/2455-5363.000007
© 2015 Ikeuchi T, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Active surveillance; Electrode; Hoechst; Dyes; Potentiostat
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important pathogens that cause nosocomial infections. However, microbiological culture techniques take a few days to yield results; therefore, a simple, cost-effective, and rapid detection system is required for screening for MRSA and related bacteria: Methicillin-resistant Staphylococcus epidermidis (MRSE) carriers during the hospital admissions process. In this study, we described the simplified method using by one-time use and screen-printed carbon electrodes, relied upon current quantification of Hoechst dyes which bound with DNA amplified via polymerase chain reaction (PCR) targeted for MRSA mecA gene. Amount of DNA-bound Hoechst molecules were measured by the hand-held potentiostat within two minutes. We found that the peak of a Hoechst-mediated current depended upon the number of MRSA isolates, and successfully distinguished between carriers and a non-carrier based on nasal swabs from the patients. This method required only 10 µL for application, and the results could be obtained within total 60 min from sample collection when a minimum of 1×103 MRSA isolates was present. These results suggested that this minimized technique has the potential to become a useful system of active surveillance for MRSA/MRSE carriers.
Methicillin-resistant Staphylococcus aureus (MRSA) has become a leading cause of infections in hospitals, and mortality from MRSA bacteremia is high [1-3]. Therefore, surveillance of patients during hospital admission for MRSA and related bacteria: Methicillin-resistant Staphylococcus epidermidis (MRSE), usually via nasal swab, is important [4,5]. Active surveillance involves the detection and tracking of patients who are asymptomatic, but carry MRSA/MRSE; moreover, compelling data support the practice of identifying carriers and using contact precautions for both carriers and infected patients to reduce spread of MRSA/MRSE within hospitals .
Usually, microbiological culture is used to identify and quantify MRSA/MRSE; however, this method is time-consuming, as bacterial growth requires overnight or a few days. Therefore, development of a highly sensitive detection system that is rapid, simple, cost-effective, and portable would be beneficial for the prevention of MRSA/MRSE transmission [4,5].
Polymerase chain reaction (PCR)-based DNA analysis has recently become routine in clinical diagnosis for the sensitive, rapid, and specific detection of viruses and bacteria . Detection of PCR-amplified genes is commonly accomplished by gel electrophoresis and/or fluorescence staining based on TaqMan chemistry. In particular, fluorescence-based detection is highly sensitive and makes quantitative real-time PCR possible . However, the apparatus for optical measurement of fluorescence intensity is not amenable to miniaturization; therefore, this technology is unsuitable for the construction of a portable system.
During studies of food preservation and food safety, we have investigated the development of electrochemical sensors for nucleic acids, including for DNA from E. coli . Electrochemical-based DNA detection systems involve relatively simple technology that is highly amenable to miniaturization; such detection systems allow for rapid, label-free DNA measurement with low power consumption [10,11].
Here, we describe development of a simplified detection system for MRSA/MRSE targeted for mecA gene that involves a screen-printed carbon electrode and a hand-held potentiostat. This electrode could efficiently detect MRSA/MRSE by measuring DNA amplification using PCR with Hoechst; dyes used to stain DNA.
Materials and Methods
Patients and samples
This study was approved by the Research Ethics Committee of Osaka University and assigned accession number 11159-6. Two patients provided written, informed consent for their participation in this study.
Upon hospital admittance, nasal swab samples were isolated from the patients, each of whom was admitted to Osaka University Hospital in Suita City, Osaka, Japan via the emergency room. Sample A was from a 37-year-old female patient who had been admitted because of severe hepatic failure that resulted from acute viral infection; sample B was from a 25-year-old female patient who had been admitted because of drug addiction. Samples A and C was isolated from the same patient, but sample C was collected five days after collection of Sample A.
MRSA strain GTC 01186 (obtained from Gifu University, Japan) and isolates isolated from the patients described above were used in this study. These MRSA was cultured in brain heart infusion (BHI) broth overnight at 30°C with reciprocal shaking at 110 strokes/ min; isolates were harvested by centrifugation at 8000 xg for 5 min at 30°C. The bacterial pellet was kept at -20°C until use. The number of bacterial isolates was calculated by plating MRSA on BD MRSA-selective agar (Beckton Dickinson, Franklin Lakes, NJ, USA). The automated identification system (MicroScan WalkAway; Siemens, Munich, Germany) was ultimately used to definitively identify the MRSA/MRSE present in the patient samples .
A primer pair, MecA1 (5’- GTAGAAATGACTGAACGTCCGATAA -3’) and MecA2 (5’- CCAATTCCACATTGTTTCGGTCTAA -3’), was used for selective amplification of the MRSA/MRSE mecA gene(13). Each 20-µL PCR mixture contained 10×Fast Buffer, 1.5 U of SpeedSTAR HS DNA Polymerase (Takara Bio. Inc. Shiga, Japan), 200 µM of each dNTP, 1.0 µM of primers, and 1 µL of diluted culture suspension; DNA was not purified from the culture suspension; the suspension was used at each of the six indicated concentrations and added directly to each reaction mixture as template. PCR was performed in a Gene Atlas 322 thermal cycler (Astec Co., Ltd., Japan). The cycling conditions included a single initial denaturation at 95°C for 2 min, followed by 40 cycles of 95°C for 5 s (denaturation) and 60°C for 18 s (annealing and extension); the total time was ~60 min. For the negative control reactions, nuclease-free water was added instead of culture suspension to the PCR mixture. After PCR, electrophoresis through a 4%-agarose gel or electrochemical measurement were used to confirm amplification of target sequences.
Electrochemical detection of PCR amplification
Hoechst 33258 [2’-(4-hydroxyphenyl)-5-(4- methyl-1-piperazinyl)-2, 5’-bi-1H-benzimidazole), H33258] (Sigma Aldrich Co., MO, USA) was used as a reporting element for electrochemical detection as previously described [9-14]. H33258 is an electro-active molecule with high affinity for nucleic acids; H33258 can intercalate into the DNA double helix. Binding of H33258 molecules to amplified DNA causes the peak current to decrease because H33258–PCR-amplified DNA complex diffuse more slowly than free H33258 to the electrode surface.
A screen-printed carbon electrode chip was used for the electrochemical measurements (Figure 1A). The carbon-based chip contained a three-electrode system that comprised a working electrode, counter electrodes, and the Ag/AgCl reference electrode [9-15]. The working electrode area was 3.04 mm2, and the total size, including the connection part and the carbon barrier that prevented solution from flowing into the chip connector, was 12.5 mm×4 mm×0.3 mm . A small volume of solution (10 µL) was measured by direct application onto the electrode surface, and each chip was discarded after a single use. The screen-printed chips were affordable, and insertion of a new chip into the connector was fast and easy [9,10]. Furthermore, each chip was discarded after one measurement; therefore, cross-contamination over multiple measurements was avoided.
- American Thoracic Society/Infectious Diseases Society of America (2005) Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia. Am J Respir Crit Care Med 171: 388-416.
- Isobe M, Uejima E, Seki M, Yamagishi Y, Miyawaki K, et al. (2012) Methicillin-resistant Staphylococcus aureus bacteremia at a university hospital in Japan. J Infect Chemother 18: 841-848.
- American Thoracic Society (1996) Hospital-acquired pneumonia in adults; diagnosis, assessment of severity, initial antimicrobial therapy, and preventative strategies. A consensus statement. Am J Respir Crit Care Med 153: 1711-1725.
- Taguchi H, Matsumoto T, Ishikawa H, Ohta S, Yukioka T (2012) Prevalence of methicillin-resistant Staphylococcus aureus based on culture and PCR in inpatients at a tertiary care center in Tokyo, Japan. J Infect Chemother 18: 630-636.
- Takahashi Y, Takesue Y, Uchino M, Ikeuchi H, Tomita N, et al. (2014) Value of pre- and postoperative meticillin-resistant Staphylococcus aureus screening in patients undergoing gastroenterological surgery. J Hosp Infect 87: 92-97.
- Ghazal SS, Hakawi AM, Demeter CV, Joseph MV, Mukahal MA (2011) Intervention to reduce the incidence of healthcare-associated methicillin-resistant Staphylococcus aureus infection in a Tertiary Care Hospital in Saudi Arabia. Infect Control Hosp Epidemiol 32: 411-413.
- Yanagihara K, Kitagawa Y, Tomonaga M, Tsukasaki K, Kohno S, et al. (2010) Evaluation of pathogen detection from clinical samples by real-time polymerase chain reaction using a sepsis pathogen DNA detection kit. Crit Care 14: R159.
- Cercenado E, Marín M, Burillo A, Martín-Rabadán P, Rivera M, et al. (2012) Rapid detection of Staphylococcus aureus in lower respiratory tract secretions from patients with suspected ventilator-associated pneumonia: evaluation of the Cepheid Xpert MRSA/SA SSTI assay. J Clin Microbiol 50: 4095-4097.
- Yamanaka K, Ikeuchi T, Saito M, Nagatani N, and Tamiya E (2012) Electrochemical detection of specific DNA and respiratory activity of Escherichia coli. Electrochimica Acta 82: 132-136.
- Nagatani N, Yamanaka K, Saito M, Koketsu R, Sasaki T, et al. (2011) Semi-real time electrochemical monitoring for influenza virus RNA by reverse transcription loop-mediated isothermal amplification using a USB powered portable potentiostat. Analyst 136: 5143-5150.
- Nagatani N, Yamanaka K, Ushijima H, Koketsu R, Sasaki T, et al. (2012) Detection of influenza virus using a lateral flow immunoassay for amplified DNA by a microfluidic RT-PCR chip. Analyst 137: 3422-3426.
- Seki M, Gotoh K, Nakamura S, Akeda Y, Yoshii T, et al. (2013) Fatal sepsis caused by an unusual Klebsiella species that was misidentified by an automated identification system. J Med Microbiol 62: 801-803.
- Jonas D, Speck M, Daschner FD, Grundmann H (2002) Rapid PCR-based identification of methicillin-resistant Staphylococcus aureus from screening swabs. J Clin Microbiol 40: 1821-1823.
- Saito M, Kobayashi M, Iwabuchi S, Morita Y, Takamura Y, et al. (2004) DNA condensation monitoring after interaction with hoechst 33258 by atomic force microscopy and fluorescence spectroscopy. J Biochem 136: 813-823.
- Yamanaka K, Saito M, Kondoh K, Hossain MM, Koketsu R, et al. (2011) Rapid detection for primary screening of influenza A virus: microfluidic RT-PCR chip and electrochemical DNA sensor. Analyst 136: 2064-2068.
- Takahashi H, Seki M, Yamamoto N, Hamaguchi S, Ojima M, et al. (2015) Usefulness of a phage-open reading frame typing (POT) kit for rapid identification of Methicillin-Resistant Staphylococcus aureus (MRSA) transmission in a tertiary hospital. Infection and Drug Resistance 8: 107-111.