scholarly journals Specific Detection of Clostridium botulinum Types A, B, E, and F Using the Polymerase Chain Reaction

2002 ◽  
Vol 85 (5) ◽  
pp. 1025-1028 ◽  
Author(s):  
Kathy E Craven ◽  
Joseph L Ferreira ◽  
Mark A Harrison ◽  
Paul Edmonds
Keyword(s):  
Polymerase Chain Reaction ◽  
Clostridium Botulinum ◽  
Extraction Procedure ◽  
Toxin Gene ◽  
Chain Reaction ◽  
Gene Target ◽  
Human Illness ◽  
Polymerase Chain ◽  
Clostridial Species ◽  
Thermal Cycler

Abstract Clostridium botulinum organisms generally produce 1 of 4 neurotoxin types (A, B, E, and F) associated with human illness. Neurotoxin type determination is important in identification of the bacterium. A polymerase chain reaction (PCR) method was developed to identify 24 h botulinal cultures as potential types A, B, E, and F neurotoxin producers as well as other clostridial species which also produce neurotoxins. Components of the PCR and amplification conditions were adjusted for optimal amplification of toxin gene target regions to enable simultaneous testing for types A, B, E, and F in separate tubes using a single thermal cycler. Each primer set was specific for its corresponding toxin type. A DNA extraction procedure was also included to remove inhibitory substances that may affect amplification. This procedure is rapid, sensitive, and specific for identification of toxigenic C. botulinum.

scholarly journals Design and development of polymerase chain reaction thermal cycler using proportional-integral temperature controller

2018 ◽  
Vol 14 (2) ◽  
pp. 213-218
Author(s):  
Chong Kim Soon ◽  
Nawoor Anusha Devi ◽  
Kok Beng Gan ◽  
Sue-Mian Then
Keyword(s):  
Polymerase Chain Reaction ◽  
Low Cost ◽  
Maximum Temperature ◽  
Gradient System ◽  
Chain Reaction ◽  
Temperature Controller ◽  
Proportional Integral ◽  
Integral Temperature ◽  
Polymerase Chain ◽  
Thermal Cycler

A thermal cycler is used to amplify segments of DNA using the polymerase chain reaction (PCR). It is an instrument that requires precise temperature control and rapid temperature changes for certain experimental protocols. However, the commercial thermal cyclers are still bulky, expensive and limited for laboratory use only.  As such it is difficult for on-site molecular screening and diagnostics. In this work, a portable and low cost thermal cycler was designed and developed. The thermal cycler block was designed to fit six microcentrifuge tubes. A Proportional-Integral temperature controller was used to control the thermal cycler block temperature. The results showed that the maximum temperature ramp rate of the developed thermal cycler was 5.5 °C/s. The proportional gain (Kp) and integral gain (Ki) of the PI controller were 15 A/V and 1.8 A/Vs respectively. Finally, the developed thermal cycler successfully amplified six DNA samples at the expected molecular weight of 150 base pair. It has been validated using the Eppendorf Mastercycler nexus gradient system and gel electrophoresis analysis

scholarly journals Investigation of Different Commercially Available Real Time-Polymerase Chain Reaction Kits for SARS-CoV-2 Diagnosis

2021 ◽  
Author(s):  
Rajeev Kumar Jain ◽  
Nagaraj Perumal ◽  
Rakesh Shrivastava ◽  
Kamlesh Kumar Ahirwar ◽  
Jaya Lalwani ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Internal Control ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Specific Gene ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Gene Target ◽  
Polymerase Chain

Introduction: The whole world is facing an ongoing global health emergency of COVID-19 disease caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is a gold standard in the detection of SARS-CoV-2 infection. Presently, many single tube multiple gene target RT-PCR kits have been developed and are commercially available for Coronavirus Disease 2019 (COVID-19) diagnosis. Aim: To evaluate the performance of seven COVID-19 RT-PCR kits (DiagSure, Meril, VIRALDTECT II, TruPCR, Q-line, Allplex and TaqPath) which are commercially available for COVID-19 RT-PCR diagnosis. Materials and Methods: This observational study was conductedat the State Virology Laboratory (SVL), Gandhi Medical College, Bhopal, Madhya Pradesh, India. Seven commercially available kits have been evaluated on the basis of: (i) number of SARS-CoV-2 specific gene target; (ii) human housekeeping genes as internal control; (iii) RT-PCR run time; and (iv) kit performances to correctly detect SARS-CoV-2 positive and negative RNA samples. A total of 50 RNA samples (left over RNA) were included, master mix preparation, template addition and RT-PCR test has been performed according to kits literature. At the end of PCR run, mean and standard deviation of obtained cut-off of all kits were calculated using Microsoft Excel. Results: All seven RT-PCR kits performed satisfactory regarding the reproducibility and they could correctly identify 30 positive and 20 negative RNA samples. RNA samples (group C) having low viral loads with a high Cycle threshold (Ct) value (>30) were also detected by all these seven kits. Obtained Ct values of each group was in parallel range in comparison with the initial testing Ct values. Kits were found to be superior which contains primers and probes for three SARS-CoV-2 specific gene targets, have human housekeeping gene as internal control and taking less time to complete RT-PCR. Conclusion: All seven COVID-19 RT-PCR kits included in this study demonstrated satisfactory performance and can be used for the routine molecular diagnosis of COVID-19 disease.

Detection of Clostridium botulinum type F using the polymerase chain reaction

1994 ◽  
Vol 8 (5) ◽  
pp. 365-373 ◽  
Author(s):  
Joseph L. Ferreira ◽  
Mostafa K. Hamdy ◽  
Steven G. McCay ◽  
Mark Hemphill ◽  
Nameer Kirma ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Clostridium Botulinum ◽  
Chain Reaction ◽  
Botulinum Type ◽  
Polymerase Chain

scholarly journals Intracellular amplification of proviral DNA in tissue sections using the polymerase chain reaction.

1992 ◽  
Vol 40 (3) ◽  
pp. 333-341 ◽  
Author(s):  
K P Chiu ◽  
S H Cohen ◽  
D W Morris ◽  
G W Jordan
Keyword(s):  
Polymerase Chain Reaction ◽  
Cultured Cells ◽  
Chain Reaction ◽  
Tissue Sections ◽  
Mouse Mammary Tumor ◽  
Tumor Virus ◽  
Polymerase Chain ◽  
Thermal Cycler ◽  
Cellular Dna

We developed a new method to amplify cell DNA in situ using the polymerase chain reaction (PCR). Proviral sequences of mouse mammary tumor virus (MMTV) contained in cultured cells and tissue sections were amplified intracellularly using a thermal cycler. Two techniques were employed to maintain the localization of the amplified DNA. First, complementary tails at the 5' ends of the oligonucleotide primers resulted in the synthesis of high molecular weight concatamers containing the target sequences. Second, the PCR was carried out in a thin film of agarose solidified over the tissue sections. The specifically amplified and localized DNA was then detected by in situ hybridization (ISH). Our results demonstrate that (a) DNA in tissue sections can serve as the target for the polymerase chain reaction in situ, (b) cell morphology is maintained, and (c) a target of 167 BP can be specifically detected in individual cells. This technique should be generally applicable to amplifying cellular DNA targets in tissue sections for detection in situ.

scholarly journals An Innovative Rapid Thermal Cycling Device for Polymerase Chain Reaction

2008 ◽  
Vol 2 (2) ◽  
Author(s):  
Shadi Mahjoob ◽  
Kambiz Vafai
Keyword(s):  
Heat Transfer ◽  
Polymerase Chain Reaction ◽  
Temperature Distribution ◽  
Thermal Cycling ◽  
Thermal Management ◽  
Uniform Temperature ◽  
Chain Reaction ◽  
Temperature Ramp ◽  
Polymerase Chain ◽  
Thermal Cycler

Polymerase chain reaction (PCR) is the most commonly used molecular biology technique to amplify nucleic acid (DNA and RNA) in vitro. This technique is highly temperature sensitive and thermal management has an important role in PCR operation in reaching the required temperature set points at each step of the process (denaturing, annealing and elongation). In this work, an innovative microfluidic PCR thermal cycling device is designed to increase the heating∕cooling thermal cycling speed while maintaining a uniform temperature distribution throughout the substrate containing the aqueous nucleic acid sample. The device design is incorporating the jet impingement and micro-channel thermal management technologies utilizing a properly arranged configuration filled with a porous medium. Porous Inserts are attractive choices in heat transfer augmentation. They provide a very large surface area for a given volume which is a key parameter in heat transfer processes. Various effective parameters that are relevant in optimizing this flexible thermal cycler are investigated such as thermal cycler configuration, thickness of inlet and exit fluid channels, fluid flow rate and velocity, the porous matrix material and properties, and utilization of thermal grease. An optimized case is established based on the effects of the cited parameters on the temperature ramp, temperature distribution and the required power for circulating the fluid in the thermal cycler. The results indicate that the heating∕cooling temperature ramp (temperature change per heating∕cooling cycling time) of the proposed device is considerably higher (150.82◻C∕s) than those in literature. In addition, the proposed PCR offers a very uniform temperature in the substrate while utilizing a low power.

Failure Prediction of Polymerase Chain Reaction Thermal Cycler

2017 ◽  
Author(s):  
Mi-So Lee ◽  
Chan-Young Park ◽  
Yu-Seop Kim ◽  
Hye-Jeong Song ◽  
Jong-Dae Kim
Keyword(s):  
Polymerase Chain Reaction ◽  
Failure Prediction ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Thermal Cycler

scholarly journals Low-Cost Temperature Logger for a Polymerase Chain Reaction Thermal Cycler

2016 ◽  
Vol 6 (11) ◽  
pp. 328 ◽  
Author(s):  
Chan-Young Park ◽  
Jae-Hyeon Cho ◽  
Yu-Seop Kim ◽  
Hye-Jeong Song ◽  
Jong-Dae Kim
Keyword(s):  
Polymerase Chain Reaction ◽  
Low Cost ◽  
Temperature Logger ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Thermal Cycler
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