Polymerase Chain Reaction Machine Research Articles

Ultrasensitive plasmonic photothermal immunomagnetic bioassay using real-time and end-point dual-readout

One of the most straightforward and sensitive methods for combining the specificity of immunological recognition with the amplification capability of polymerase chain reaction (PCR) is the fluorescence-based immuno-PCR. But up until now, the majority of PCR-based immunoassays have utilized the pricey and lengthy Peltier-based heating technique. Herein, we introduce a plasmonic photothermal quantitative immuno-PCR (PPT-qiPCR) assay with real-time and end-point dual readout for biomarker detection using multifunctional plasmonic immunomagnetic nanoparticles (PIMNs). PPT-qiPCR is simple, time-saving and inexpensive compared with conventional iPCR platforms using PCR machine. We showed that an immunomagnetic technique utilizing PIMNs can shorten the time needed for collection, washing, and concentrating target proteins in the enzyme-linked immunosorbent assay (ELISA) procedure. Real-time readout also permits quick preliminary discrimination of target proteins with apparent sensitivity in commercial ELISA investigations. Additionally, in-situ end-point fluorescence or colorimetric detection using only the human eye considerably improve the assay sensitivity. Our PPT-qiPCR can significantly advance next-generation point-of-care molecular diagnostic technology and is anticipated to be immediately useful for the sensitive and quick identification of several disease biomarkers.

Wireless Temperature Measurement Validation Method for PCR Machines by Magnetic Hall-Effect Sensor

The temperature validation controlled by temperature indication has a vital role in the polymerase chain reaction (PCR) test machine or thermal cycler. However, the validation process is complicated for several types of thermal cycler. Some PCR test machines must close the lid tightly while running. It makes the probe’s cable of the temperature sensor might be pinched or break when the thermal cycler lid is closed. Opening the lid (open-air condition) makes the measurement will not accurate. To solve this problem, wireless temperature measurement and validation methods for PCR machines are developed based on magnetic field measurements. The magnetic field of the object will respond to any changes in temperature. The hall-effect sensor, which is validated by gauss meter, detects any magnetic response a certain material covers even the object. This detection yields output data processed to find the thermal cycler's appropriate temperature wireless validation method. The experiment used a Neodymium magnet as a wireless probe. The position of the Neodymium magnet pole significantly affected the relation between magnetic flux and temperature in experimental results. The reversed pole toward sensors had better linearity (R2= 0.8062) than the unreversed pole (R2= 0.7794). The annealing step commonly achieved the optimum measurement uncertainty. However, the measurement uncertainty and signal sensitivity investigation recommended employing the beneficial combination of pole magnet position to design the temperature validator based on magnetic induction for a closed lid thermal cycler (PCR machine). Overall, the experimental yields can be used to build a wireless temperature validator for a sealed PCR machine based on magnetic induction.

An Interdisciplinary Design Project for Undergraduate Engineering Training – Portable Real-Time Polymerase Chain Reaction System

Our world has been permanently changed by the pandemic outbreak of COVID-19 starts around the end of 2019. In the first few months of 2020, the whole world was in urgent need of an effective, easy, and quick method for the identification of the infection of the new virus. Polymerase Chain Reaction (PCR) machine, which can test DNA samples by rapidly making millions of copies of a specific DNA sample through the PCR process, including the COVID-19 virus, can perfectly fit this demand. In this study, a design project on PCR is introduced for undergraduate education in electrical and mechanical engineering. The objective of this project is to develop a low-cost, ease-of-use, wallet-size, portable real-time PCR (RT-PCR) machine for accurate testing of various bacteria or viruses. The key function of the PT-PCR system is to precisely control and maintain the temperature of the biosample solution within a range between 55°C and 95°C. This project provides students opportunities in studying and practicing a wide range of engineering technics and skills, including mechanical design, electronics design, microcomputer programming, data acquisition and processing, etc. Students can gain comprehensive understanding of the design of multiphysics system after they overcome various challenges emerging in the project. From the view of engineering education, the process of this project development has demonstrated the importance and benefits of adopting complex interdisciplinary engineering problems for student teams to solve, especially those involve contemporary issues.

Laboratory Surge Response to the COVID-19 Pandemic: An Incident Command System Approach.

The COVID-19 pandemic has reaffirmed the critical significance of effective diagnostics in outbreak response. In Taiwan, the COVID-19 wave in May 2021 led to a rapidly growing demand for SARS-CoV-2 diagnostic tests. To meet the challenge, an extensive system-wide emergency preparedness plan, hospital emergency incident command system (HEICS), was developed to deal with emergencies involving healthcare systems. During the wave of the COVID-19 outbreak, a 19.4-fold increase in SARS-CoV-2 PCR (polymerase chain reaction) diagnostic tests occurred in the hospital. The incident commander of TCVGH reviewed COVID-19 related events daily and purchased a high-throughput PCR machine for SARS-CoV-2 PCR diagnostic tests. In addition, the Department of Operations was responsible for staff scheduling and educational training. The turn-around times of SARS-CoV-2 diagnostic tests were shortened from 21.2 hours to 5.8 hours in the second week of the COVID-19 wave. Implementation of HEICS integrated resources could be helpful for expanding surge capacity during future outbreaks.

Heating and Cooling Rate Study on Water Cooling Thermal Cycler using Aluminium Block Sample

Temperature measurement has many applications in medical devices. In recent days, body temperature become the main screening procedure to justify people infected by SARS-CoV-2. Related to pandemic situation due to SARS-Cov-2, Polymerase Chain Reaction (PCR) method become the most accurate and reliable detection method. This method employs a device named PCR machine or Thermal Cycler. In this research, we focus to build a Thermal Cycler using a low-cost material such as aluminium and using a liquid coolant as the cooling system. We use 2 types of coolant solution: mineral water and generic liquid coolant. Peltier device in thermal cycler serves as heating and cooling element. In heating rate experiments, generic liquid coolant shows a better result than using mineral water due to specific heat capacity and thermal conductivity of water. In the cooling rate experiments, the water pump is activated to stream the liquid solution, the flow rate of liquid solution is influenced by viscosity of the liquid. Generic liquid coolant has approx. 4,5 times greater viscosity than water. The higher flow rate means better performance for cooling rate. Using 2 pieces of 60-Watt heaters and a 60-Watt chiller and aluminium material as block sample, our research shows a heating and cooling rate up to approx. 0,1°C/s. Compared to commercially thermal cycler, our thermal cycler has a lower wattage; this lower wattage performance has been tradeoff with lower ramping rate. Some factors are suspected become the source of contributors of lower ramping rate.

Magnetic Multiplex Loop Mediated Isothermal Amplification (MM-LAMP) technique for simultaneous detection of dengue and chikungunya virus

Dengue and chikungunya viruses are arthropod borne virus spread through common vector instigating infection in human. There has been an increased recognition that more attention needs to be paid to similar sympotoms caused by both of the virus as they spread in the same region at same time. It warrants need of cost effective, user friendly and rapid multiplex diagnostic technique which could simultaneously diagnose and identify between two virus diseases in resource poor setting. A magnetic multiplex loop mediated isothermal amplification (MM-LAMP) technique was developed by coupling multiplex LAMP with magnetic particle-based naked eye visualization to overcome the shortcoming of simultaneous detection of both diseases. In recent years this technology has emerged as a particularly attractive candidate as amplification reaction process completes within 45 min. The first step involves multiplexing biotin and digoxigenin coated dengue and chikungunya primers respectively in LAMP reaction followed by precipitation of the amplified DNA with polyethylene glycol (PEG) buffer and finally clumping with streptavidin and anti-digoxigenin coated magnetic particle for virus discrimination and naked eye visualization. The DNA detection limit of MM LAMP visualization was 51.65 ng/μl which is comparable to the electrophoresis base UV light visualization. The results showed potential superiority over standard methods polymerase chain reaction (PCR). This current advancement empowers multiplex LAMP utility in resource limited setting without using any of the florescent dyes, turbidimeter, or the sophisticated quantitative PCR machine etc which restrict multiplex LAMP technique to laboratorial use only. We have proposed a novel method without such limitations. This technique has potential as a point of care technique for simultaneous detection of two diseases.

Assessing standards for prevention of early onset group B streptococcal (GBS) disease in Ireland

Background:Early onset group B streptococcal (GBS) disease can cause significant neonatal morbidity and mortality. There is currently no Irish national guideline for GBS screening, and protocols vary across maternity units. Polymerase chain reaction (PCR) testing at induction or labour onset informs triage for antibiotic prophylaxis; however, there are human and infrastructural resource requirements to enable widespread implementation.Aim:Our aim was to identify current standard practices for GBS prevention in Irish obstetric and neonatal services and to utilise this data to inform the need for, and potential impact of implementation of, a national guideline.Methods:A questionnaire on GBS screening, management and existing resources was completed by an informed staff member from each of the 19 Irish maternity units, including questions regarding timing and method of screening, antibiotic usage, and neonatal management.Results:One unit (5.2%) performs routine GBS screening at 35–37 weeks of gestation. Twelve units (63%) screen for GBS following spontaneous rupture of membranes (SROM) after 37 weeks, of which two (17%) perform PCR and ten (83%) culture testing. Seventeen units (89.3%) have access to a GeneXpert PCR machine, and of these, two (11.7%) use the machine for rapid GBS testing. Two units screen patients for GBS at either the start of labour or induction of labour. Four units (21%) use the neonatal early onset sepsis (EOS) calculator. Sixteen units (84%) do not treat asymptomatic infants born to GBS-positive mothers. Conclusion:There is a lack of consistency in the methods for GBS screening and disease prevention across the country, highlighting the need for a national guideline accompanied by an implementation plan and budget to standardise care.

Amplifikasi DNA Kandidat Gen Kuda Pacu Sumba

The Sumba horse is one of the local horses in Indonesia which is known as racing horse Several candidate genes are known to influence the outward characteristics of the Sumba racehorse, which play main role in the development of the horse's muscles from embryo to adulthood. This research aims to identify candidate genes for the Sumba racehorse in stallion and mares. Blood samples from 5 stallions and 5 mares were collected and analyzed. The method used in this research was by using polymerase chain reaction (PCR), electrophoresis and DNA sequencing. The results of DNA amplification fragments at a temperature of 600c showed a fragment size of 463 bp. A total of 10 samples were sequenced on the PCR machine. The forward primer was 5'-TATTCTTCTTGGGAGGGAGGACTACT-3 'and reverse primer was 5'-GCAAGTAATTAGCACAAAAATTTGAATG-3'. The obtained data was analyzed using the Basic Local Alignment Sealing Tool (BLAST). Result of this study could be used as an initial identification of candidate genes for racing activity in stallion and mares that can complement the selection of racing horses.

Determining the feasibility of constructing a real-time polymerase chain reaction instrument locally: The cost of intellectual property rights and research and development

BACKGROUND: Real-time polymerase chain reaction (PCR) machines have advanced the field of microbiology by introducing a robust method for high-throughput detection of nucleic acid targets. Unfortunately, these instruments are quite expensive to purchase and maintain, despite containing hardware parts that are relatively inexpensive. AIMS AND OBJECTIVES: We believe that it may be possible to mitigate costs in low- and middleincome countries (LMICs) by building such a PCR instrument from assembled parts. However, the major hindrance is the cost of intellectual property rights (IPRs), associated with research and development (R and D). The aim of this project was to understand the hardware costs, IPR royalties, profit margins, and the cost of R and D associated with PCR instrument manufacturing. MATERIALS AND METHODS: We conducted a review of two PCR machines to examine the differences in their technical and hardware specifications and quantify the cost of assembled parts alone. We also ran a parallel assay using the two machines to understand how the differences in technologies affected assay results. RESULTS AND CONCLUSION: Based on our analysis, we found that assembling a PCR machine using assembled parts can make these instruments more affordable in LMICs. There is a need to have collaboration and agreement between machine licensing, patent, and copyright holders to facilitate this process in LMICs.

Machine-Free Polymerase Chain Reaction with Triangular Gold and Silver Nanoparticles.

Photothermal effects of metal nanoparticles (NPs) are used for various biotechnological applications. Although NPs have been used in a polymerase chain reaction (PCR), the effects of shape on the photothermal properties and its efficiency on PCR are less explored. The present study reports the synthesis of triangular gold and silver NPs, which can attain temperatures up to ∼90 °C upon irradiation with 808 nm laser. This photothermal property of synthesized nanoparticles was evaluated using various concentrations, irradiation time, and power to create a temperature profile required for variable-temperature PCR. This study reports a cost-effective, machine-free PCR using both gold and silver triangular NPs, with efficiency similar to that of a commercial PCR machine. Interestingly, addition of triangular NPs increases PCR efficiency in commercial PCR reactions. The higher PCR efficiencies are due to the direct binding and unfolding of double-stranded DNA as suggested by circular dichroism and UV spectroscopy. These findings suggest that triangular NPs can be used to develop cost-effective, robust machine-free PCR modules and can be used in various other photothermal applications.

Multiplex Solid-Phase Real-Time Polymerase Chain Reaction without DNA Extraction: A Rapid Intraoperative Diagnosis Using Microvolumes

Current polymerase chain reaction (PCR) methods for the diagnosis of infections are time consuming and require large sample volume and skilled technicians. We developed a novel, easy-to-use, and rapid (processing time, 1 minute; total time, 33 minutes) multiplex real-time PCR test (Direct Strip PCR) that did not require DNA extraction to detect 9 pathogens that could cause uveitis in 20-μl samples. Multicenter prospective evaluation of a diagnostic PCR test. A total of 511 participants (patients with infectious uveitis and controls) were examined at 18 institutes worldwide. After validation, intraocular fluid samples were subjected to etiologic or exclusive diagnosis, including intraoperative rapid diagnosis. The concordance and correlations between Direct Strip PCR and quantitative PCR (qPCR) results. Direct Strip PCR exhibited rapid detection, good repeatability and specificity, long storage stability, and detection ability equal to that of qPCR. It also showed low interinstitutional variability compared with qPCR, even when PCR beginners used various real-time PCR machines. The Direct Strip PCR for 9 pathogens exhibited high concordance against the qPCR (positive concordance rate, 98.8%-100%; negative concordance rate, 99.8%-100%; κ coefficient, 0.969-1.000; P < 0.001-0.031). Additionally, results obtained using Direct Strip PCR and qPCR were highly correlated (ρ= 0.748; P < 0.001). This assay was used for rapid intraoperative diagnosis. The Direct Strip PCR test may improve the prognosis of various infectious diseases because it facilitates rapid etiologic evaluation at the first hospital visit and can be used for intraoperative diagnosis.

Rapid Detection of Wheat Blast Pathogen Magnaporthe oryzae Triticum Pathotype Using Genome-Specific Primers and Cas12a-mediated Technology

Wheat blast, caused by the fungus Magnaporthe oryzae Triticum (MoT) pathotype, is a devastating disease persistent in South America and Bangladesh. Since MoT generally fails to cause visual symptoms in wheat until the heading stage when the infection would have advanced, disease control by fungicide application solely based on the detection of visual symptoms is ineffective. To develop an accurate and sensitive method to detect MoT at the seedling and vegetative stages for disease control, we sequenced the genomes of two MoT isolates from Brazil and identified two DNA fragments, MoT-6098 and MoT-6099, that are present in the MoT genome but not in the genome of the rice-infecting Magnaporthe oryzae Oryzae (MoO) pathotype. Using polymerase chain reaction (PCR), we confirmed the specificity of the two markers in 53 MoT and MoO isolates from South America and Bangladesh. To test the efficiency of the two markers, we first established a loop-mediated isothermal amplification (LAMP) method to detect MoT at isothermal conditions, without the use of a PCR machine. Following this, we used the Cas12a protein and guide RNAs (gRNAs) to target the MoT-6098 and MoT-6099 sequences. The activated Cas12a showed indiscriminate single-stranded deoxyribonuclease (ssDNase) activity. We then combined target-dependent Cas12a ssDNase activation with recombinase polymerase amplification (RPA) and nucleic acid lateral flow immunoassay (NALFIA) to develop a method that accurately, sensitively, and cost-effectively detects MoT-specific DNA sequences in infected wheat plants. This novel technique can be easily adapted for the rapid detection of wheat blast and other important plant diseases in the field.

Rapid, large-scale, and effective detection of COVID-19 via non-adaptive testing

Pooling of samples can increase lab capacity when using Polymerase chain reaction (PCR) to detect diseases such as COVID-19. However, pool testing is typically performed via an adaptive testing strategy which requires a feedback loop in the lab and at least two PCR runs to confirm positive results. This can cost precious time. We discuss a non-adaptive testing method where each sample is distributed in a prescribed manner over several pools, and which yields reliable results after one round of testing. More precisely, assuming knowledge about the overall incidence rate, we calculate explicit error bounds on the number of false positives which scale favourably with pool size and sample multiplicity. This allows for hugely streamlined PCR testing and cuts in detection times for a large-scale testing scenario. A viable consequence of this method could be real-time screening of entire communities, frontline healthcare workers and international flight passengers, for example, using the PCR machines currently in operation.

Small-Size Polymerase Chain Reaction Device with Improved Heat Transfer and Combined Feedforward/Feedback Control Strategy

We report improvements on the heat transfer and on the control strategy of a thermal cycler implemented on a novel device for performing a fast polymerase chain reaction (PCR). The reduction of the thermal mass of the sample holder and the direct contact with the sample allow for a significant reduction of the transition times, while the hybrid feedforward/feedback controller can rely on inexpensive components (actuators, sensors, processor) and still achieve minimal over/undershooting and good temperature stability. The design of the device has been improved by performing transient heat conduction analysis on the highly heat-conductive sample holder and on the solid metal body of the device which rapidly dissipates the excess heat produced during the thermal cycling. In the current setup, the sample holder hosts nine 1 μL samples covered with mineral oil, which can be simultaneously read in real time by the detection module designed in-house and installed on the device. An increase in speed of the PCR amplification was achieved with a reduction of the transition time of 63.8% when compared against a commercial real-time PCR machine. Our work shows that a complete, stand-alone, and ready-to-use quantitative PCR instrument can be fabricated from inexpensive and easily available components and it can achieve fast thermal cycling thanks to a hybrid control strategy.

Polymerase Chain Reaction using "V" Shape Thermal Cycling Program.

Polymerase chain reaction (PCR) is the most commonly used technique in molecular biology and diagnostics. To achieve faster PCR reaction time, two strategies were employed by previous studies. That includes improving the thermal ramp rate by developing novel devices to reduce the time wasted on temperature transitions and cutting the holding time in every step, which could even lead to compromise in amplification efficiency. Hence the need to further improve the technique.Methods: A different way to achieve fast DNA amplification is developed by using the previously thought wasted time spent on heating and cooling the samples to finish the amplification. That means the holding time of the three procedures are omitted and this could be carried out on the ordinary PCR thermal cyclers.Results: 2/3 of the amplification time is easily saved, compared to the conventionally used method. Additionally, the reaction time could be further reduced by using longer primers with higher melting temperature (Tm). The record time of the “V” shape Polymerase chain reaction (VPCR) conducted on ordinary PCR machine for amplification of a 98 bp fragment is 8 min. Furthermore, VPCR still retains the merits of traditional PCR technique, including specificity, sensitivity, generality, and compatibility with quantitative detection.Conclusion: It is confirmed that the three procedures of PCR could be completed during the dynamic heating and cooling process when the cyclers are run at a moderate thermal ramp rate. As VPCR described here is based on the current PCR system, it could be implemented in any biological Lab immediately and provide great convenience to the people working in the field of life science and human health.

Analysis and Normalization of Real-Time Polymerase Chain Reaction (PCR) Experimental Data.

In real-time polymerase chain reaction (PCR), also called quantitative real-time PCR [or simply quantitative PCR (qPCR)] or kinetic PCR, the amplification of DNA is monitored by the detection and quantitation of a fluorescent reporter signal, which increases in direct proportion to the amount of PCR product in the reaction. The fluorescent reporter is excited by light from the real-time PCR machine, a fluorescence-detecting thermocycler. By recording the amount of fluorescence emission at each cycle, the PCR can be monitored during the exponential phase when the first significant increase in the amount of PCR product correlates with the initial amount of target template. The ability to quantify the amplified DNA during the exponential phase of the PCR, when none of the components of the reaction is limiting, has resulted in dramatically improved precision in the quantitation of target sequences. In addition, because of the high sensitivity of fluorometric detection, real-time PCR is capable of measuring the initial concentration of target DNA over a vast dynamic range (up to eight or nine orders of magnitude) and with a high degree of sensitivity (as little as one copy of template DNA). Although it is a powerful technique, researchers often face challenges in reliability and reproducibility because of the lack of assay standardization. Therefore, it is critical to optimize the reagents and reaction conditions, include proper internal and external controls, and perform rigorous data analysis in order to generate accurate and reproducible results in real-time PCR experiments.

Development and Manufacturing of World Grade Programmable Thermal Cyclers for Polymerase Chain Reaction in Pakistan: A Case of Biomedical Engineering

Manufacturing is the backbone of a modern state and has a direct impact on life style of a common person of a society. We present here the development and manufacturing of Polymerase Chain Reaction (PCR) machines by the Pakistan Atomic Energy Commission (PAEC), Pakistan. Steps of the development and manufacturing of PCR machines are explained. Important design features of MERADD PCR machines and their implementation are documented here. Results of the MERADD PCR machine are compared with those of competitive imported machines. MERADD is the sole manufacturer of PCR machines in Pakistan. Quality assurance and cost analysis are also given. MERADD PCR machines produce as good results as by the imported machines, but cost is significantly lower. This paper is useful for a broad community of developers/manufacturers of medical devices and biomedical engineering in developing countries around the world.

Mitigating polymerase chain reaction/amplicon contamination in a high-risk high-burden mycobacterial reference laboratory in a resource-limited setting.

Nucleic acid amplification techniques have become important machineries in the diagnosis of several diseases in clinical laboratories. Polymerase chain reaction (PCR) contamination/amplicon contamination leading to false positivity remains a major concern in these laboratories. Prevention of these contaminations in establishing these molecular biology laboratories has been very crucial over the years. Although closed system PCRs have substantial reduction in the PCR contamination rates, the conventional probe-based hybridization methods continue to show occurrence of contamination for various reasons. The study involved checking the crucial parameters as well as the probable candidates of causing the contamination at a high-burden setting. Bringing out the most effective interventions in controlling the PCR contaminations for future endeavors stood as priority. The study explored the efficacies of different sets of interventions contributed to the process of reducing the contaminants. The detection of the contaminating PCR products or amplicons or contaminating organism is done by the genotype MTBDR plus V2 kits (Hains Life Sciences) based on DNA strip technology. The pre- and post-cleaning as well as cleaning of the working surfaces was able to bring down the mean contamination percentage by 36.5%. The combined effect of cleaning the work surfaces, the automated pipetting devices, and the AC machines was able to bring down the mean contamination percentage to 53.5% reducing the contamination rate nearly to 94.6%. Regularly cleaning the work surfaces, the automated pipetting devices, the PCR machine, and the AC machines along with it filters and exposure of UV rays significantly lowers down the mean contamination percentage.

Rapid detection of the New Delhi metallo-b-lactamase 1 (NDM-1) gene by loop-mediated isothermal amplification (LAMP).

New Delhi Metallo-b-lactamase (NDM-1) is an enzyme emerging around the world conferring resistance to a wide range of β-lactams agents and whose early detection is extremely important. We proposed to standardize the detection of the blaNDM-1 gene using the LOOP-mediated isothermal amplification technique (LAMP). In all, 14 Gram-negative bacterial strains isolated from patients presenting pneumonia associated with mechanical ventilation were used for the blaNDM-1 standardization by LAMP. Klebsiella pneumoniae ATCC BAA-2473 and two clinical strains were used as a positive control. All results were compared to the reaction in polymerase chain reaction (PCR), considered gold standard for this detection. There was an excellent correlation between the two techniques employed, since all measured clinical strains were negative in both employed tests and two clinical, and a reference strains were positive. The lamp technique seems to be an excellent option for the rapid detection of blaNDM-1. The amplification time is much shorter than other molecular techniques, the PCR machine is not necessary, it is easy of implementation and costs is low.

Accuracy of polymerase chain reaction-restriction fragment length polymorphism for RET rs2435357 genotyping as Hirschsprung risk

BackgroundRecently, the common RET rs2435357 variant has been shown to be strongly related to Hirschsprung disease (HSCR) in the Indonesian population. This association study was conducted in developed areas using high-throughput TaqMan polymerase chain reaction (PCR) assay. Although the TaqMan method is less time-consuming, it requires a special more expensive PCR machine and a highly skilled analyst. In this study, we analyzed the usefulness of the PCR-restriction fragment length polymorphism (RFLP) method for genotyping RET rs2435357 polymorphism in Indonesian HSCR patients given the limitation of resource allocation for more expensive technologies. Materials and methodsWe compared our previous genotyping results of RET rs2435357 in 53 HSCR patients and 86 controls using the TaqMan PCR assay with the PCR-RFLP technique. Furthermore, we included an additional 40 HSCR patients and 50 controls and subsequently genotyped all subjects using the PCR-RFLP method. ResultsCompared with our previous genotyping data of RET rs2435357 using the TaqMan PCR assay, the PCR-RFLP method indicated 100% concordant results. The overall accuracy of the PCR-RFLP for RET rs2435357 genotyping was 100%. In addition, case-control analysis demonstrated that RET rs2435357 is significantly correlated with HSCR (P = 2.2 × 10−13) with an odds ratio of 5.1 (95% confidence interval = 3.2-8.1). The transmission disequilibrium test revealed that risk allele (T) at rs2435357 is significantly overtransmitted to probands at a transmission rate (τ) of 0.87 (P = 1.5 × 10−6). ConclusionsThe PCR-RFLP method is reliable and affordable for genotyping of RET rs2435357 polymorphism in developing countries. Our results strengthen the proof that the RET rs2435357 variant is a genetic risk for HSCR in Indonesia.