A Pipeline-Based Oil-Bath PCR Method for Bacteria Detection

Abstract

Polymerase chain reaction (PCR) technology is an essential technology for point-of-care testing (POCT) application. To realize the simple, portable and fast detection of bacteria has always been an important technology. In this paper, we developed a pipeline-based oil-bath PCR method, to achieve the PCR amplification and detection for bacteria. We assembled a small-sized system with a uniform temperature environment and a stable heating module. By comparing the PCR performance with the other two existing pipeline-based methods on the reactions of both DNA fragment of H7N9 avian influenza and Escherichia Coli, the results show that the pipeline-based oil-bath PCR method is fully capable of the amplification and detection of both DNA fragment and bacteria. At the meanwhile, we performed two sets of tests, the experiment of pipeline diameter change and bacterial concentration gradient, to verify the sensitivity and universality of this method. The results show that the pipeline-based oil-bath PCR method developed by us not only gets rid of the complicated preparations, offers advantages of simple operation, low-cost and user-friendly, but also provides a stable and portable foundation for our later work.

SERS Sensors Based on Aptamer-Gated Mesoporous Silica Nanoparticles for Quantitative Detection of Staphylococcus aureus with Signal Molecular Release

“…So far, several methods such as plate culture method, enzyme-linked immunosorbent assay, molecular biological detection, for example, polymerase chain reaction, have been focused on the determination of foodborne pathogens in different matrices. These methods not only require expensive instruments and highly skilled workers but also involve intricate sample pretreatments and are time-consuming, − hence limiting their application in food safety and security.…”

Three-Dimensional Surface-Enhanced Raman Scattering Platform with Hotspots Built by a Nano-mower for Rapid Detection of MRSA

“…Bacterial pathogens have a serious impact on global public health security because they can spread through a variety of vectors, including bodily fluids, food, and water, resulting in the infection of many people. − The complications caused by bacterial pathogen infections seriously threaten human health. The traditional “gold standards” for bacterial detection, such as ELISA, PCR, and plate counting are time-consuming, which seriously limits their application in bacterial detection. , Compared with other bacteria, methicillin-resistant Staphylococcus aureus (MRSA) has become one of the greatest threats to human life because of its strong drug resistance, wide distribution, and high infection rate. − Therefore, designing a fast and sensitive MRSA detection method is very important for clinical diagnosis and treatment.…”

“…1−3 The complications caused by bacterial pathogen infections seriously threaten human health. The traditional "gold standards" for bacterial detection, such as ELISA, 4 PCR, 5 and plate counting 6 are time-consuming, which seriously limits their application in bacterial detection. 7,8 Compared with other bacteria, methicillin-resistant Staphylococcus aureus (MRSA) has become one of the greatest threats to human life because of its strong drug resistance, wide distribution, and high infection rate.…”

Construction of Very Low-Cost Loop Polymerase Chain Reaction System Based on Proportional-Integral-Derivative Temperature Control Optimization Algorithm and Its Application in Gene Detection

“…Polymerase chain reaction (PCR) technology developed in 1983 by Kary Mullis has been a well-known method and has been used widely in biological and medical invention. − By applying this technology, the natural replication process of DNA and specific DNA molecular fragment amplification in vitro could be obtained, which has been popularized and applied in the detection of various pathogenic microorganisms and parasites, especially in the point-of-care testing (POCT), such as disaster rescue, , quarantine, , and plant disease monitoring. , There exist three stages in a PCR system, namely, denaturation, anneal, and extension. , When the reagent is heated up to approximately 95 °C, the double-stranded DNA would be dissociated into a single one and combined with the primer in the next round of reactions, which is called as the denaturation of the template DNA stage. While the system’s temperature dropped to presumably 55 °C, the template of the single-strand DNA combined with the primer based on the principle of complementary base pairing, which is called as the annealing of the template DNA and the primer stage.…”

“…Polymerase chain reaction (PCR) technology developed in 1983 by Kary Mullis has been a well-known method and has been used widely in biological and medical invention. 1 − 3 By applying this technology, the natural replication process of DNA and specific DNA molecular fragment amplification in vitro could be obtained, which has been popularized and applied in the detection of various pathogenic microorganisms and parasites, 4 especially in the point-of-care testing (POCT), such as disaster rescue, 5 , 6 quarantine, 7 , 8 and plant disease monitoring. 9 , 10 There exist three stages in a PCR system, namely, denaturation, anneal, and extension.…”