Simple Polymerase Chain Reaction Research Articles

A simple and cost-effective real-time PCR method using diluted and heat-treated whole blood lysate

Although DNA extraction is a crucial step before polymerase chain reaction (PCR), attempts to perform PCR without DNA isolation have been ongoing since the 1990s. While partial success has been achieved with direct conventional PCR, the DNA isolation step has remained indispensable for real-time PCR. Here, we developed a method that does not require complete DNA isolation by lysing EDTA-treated whole blood samples through the application of osmotic pressure and heat, followed by centrifugation to get a clear lysate. Blood samples were mixed with distilled water, incubated at 95 °C for 20 min before centrifugation at 14,000 rpm for 5 min. The resulting lysates were used as templates for real-time PCR. Real-time PCRs were performed under identical conditions with lysates and DNA samples using 9 different primer sets. Target genes were successfully amplified using 1:10 and 1:5 diluted blood lysates both at 60 °C and 61 °C. The PCR efficiency for ACTB and PIK3CA differed by 20% and 14%, respectively, between the DNA samples and blood lysates. We successfully amplified all selected genes using “GG-RT PCR”, greater temperature, greater speed method, without the need for additional enzymes or buffers. GG-RT PCR presents a cost effective option for applications such as SNP analysis and deletion detection if appropriate primer designs are made.

A simple PCR‐based method for detecting Anagyrus lopezi (Hymenoptera: Encyrtidae) and Prochiloneurus pulchellus (Hymenoptera: Encyrtidae), primary and hyper parasitoids of the cassava mealybug Phenacoccus manihoti (Hemiptera: Pseudococcidae)

AbstractEstimating parasitism rates in the field is essential for developing and evaluating biocontrol strategies using parasitoids. In this study, we developed a simple polymerase chain reaction (PCR)‐based method for detecting parasitism of the cassava mealybug Phenacoccus manihoti Matile‐Ferrero (Hemiptera: Pseudococcidae) by the primary parasitoid Anagyrus lopezi De Santis (Hymenoptera: Encyrtidae) and its hyperparasitoid Prochiloneurus pulchellus Silvestri (Hymenoptera: Encyrtidae). Primers were designed to amplify partial cytochrome c oxidase subunit I genes of each species, and their sensitivity was evaluated with mealybugs that had been parasitized by A. lopezi 0, 3, and 6 days earlier, and mummified mealybugs containing A. lopezi pupae that had been parasitized by P. pulchellus 0, 3, 6, 9, and 12 days earlier. The detection rate of parasitism by A. lopezi was 100% for all ages of A. lopezi. The detection rate of parasitism by P. pulchellus ranged from 94.1% to 100%, depending on its developmental stage. For P. pulchellus, template DNA was diluted 10 times before PCR because PCR with the original concentration showed low detection rates, presumably due to the presence of PCR inhibitors. Overall, our primers can be considered sufficiently sensitive to be used for detecting each species.

Development of a multiplex polymerase chain reaction assay for detection of hepatitis C virus, hepatitis B virus, and human immunodeficiency virus 1.

Hepatitis C virus (HCV), hepatitis B virus (HBV), and human immunodeficiency virus 1 (HIV-1) are the most epidemic blood-borne viruses, posing threats to human health and causing economic losses to nations for combating the infection transmission. The diagnostic methodologies that depend on the detection of viral nucleic acids are much more expensive, but they are more accurate than serological testing. To develop a rapid, cost-effective, and accurate diagnostic multiplex polymerase chain reaction (PCR) assay for simultaneous detection of HCV, HBV, and HIV-1. The design of the proposed PCR assay targets the amplification of a short conserved region featured with a distinguishable melting profile and electrophoretic molecular weight inside each viral genome. Therefore, this diagnostic method will be appropriate for application in both conventional (combined with electrophoresis) and real-time PCR facilities. Confirmatory in silico investigations were conducted to prove the capability of the approached PCR assay to detect variants of each virus. Then, Egyptian isolates of each virus were subjected to the wet lab examination using the given diagnostic assay. The in silico investigations confirmed that the PCR primers can match many viral variants in a multiplex PCR assay. The wet lab experiment proved the efficiency of the assay in distinguishing each viral type through high-resolution melting analysis. Compared to related published assays, the proposed assay in the current study is more sensitive and competitive with many expensive PCR assays. This study provides a simple, cost-effective, and sensitive diagnostic PCR assay facilitating the detection of the most epidemic blood-borne viruses; this makes the proposed assay promising to be substitutive for the mistakable and cheap serological-based assays.

A rapid and low-cost platform for detection of bacterial based on microchamber PCR microfluidic chip.

Polymerase chain reaction (PCR) has been considered as the gold standard for detecting nucleic acids. The simple PCR system is of great significance for medical applications in remote areas, especially for the developing countries. Herein, we proposed a low-cost self-assembled platform for microchamber PCR. The working principle is rotating the chamber PCR microfluidic chip between two heaters with fixed temperature to solve the problem of low temperature variation rate. The system consists of two temperature controllers, a screw slide rail, a chamber array microfluidic chip and a self-built software. Such a system can be constructed at a cost of about US$60. The micro chamber PCR can be finished by rotating the microfluidic chip between two heaters with fixed temperature. Results demonstrated that the sensitivity of the temperature controller is 0.1℃. The relative error of the duration for the microfluidic chip was 0.02s. Finally, we successfully finished amplification of the target gene of Porphyromonas gingivalis in the chamber PCR microfluidic chip within 35min and on-site detection of its PCR products by fluorescence. The chip consisted of 3200 cylindrical chambers. The volume of reagent in each volume is as low as 0.628 nL. This work provides an effective method to reduce the amplification time required for micro chamber PCR.

Efficient PCR-based gene targeting in isolates of the nonconventional yeast Debaryomyces hansenii.

Debaryomyces hansenii is a yeast with considerable biotechnological potential as an osmotolerant, stress-tolerant oleaginous microbe. However, targeted genome modification tools are limited and require a strain with auxotrophic markers. Gene targeting by homologous recombination has been reported to be inefficient, but here we describe a set of reagents and a method that allows gene targeting at high efficiency in wild-type isolates. It uses a simple polymerase chain reaction (PCR)-based amplification that extends a completely heterologous selectable marker with 50 bp flanks identical to the target site in the genome. Transformants integrate the PCR product through homologous recombination at high frequency (>75%). We illustrate the potential of this method by disrupting genes at high efficiency and by expressing a heterologous protein from a safe chromosomal harbour site. These methods should stimulate and facilitate further analysis of D. hansenii strains and open the way to engineer strains for biotechnology.

Diagnosis of mucormycosis using a simple duplex PCR assay: Analysis of 160 clinical samples from COVID-19 patients.

Early diagnosis of mucormycosis, a severe and potentially fatal complication in immunocompromised and COVID-19 patients, is crucial for initiating timely antifungal therapy and reducing infection mortality. In this study, the diagnostic performance of a duplex polymerase chain reaction (PCR) assay was evaluated to detect Mucorales-specific and Rhizopus oryzae-specific targets in 160 clinical samples collected from 112 COVID-19 patients suspected of invasive fungal rhinosinusitis (IFRS). During potassium hydroxide (KOH) direct microscopy, non-septate hyphae were observed in 73 out of 160 samples (45.63%); however, using duplex PCR, 82 out of 160 specimens (51.25%) tested positive. Among the positive PCR samples, 67 (81.71%) exhibited a double band (both 175 and 450 base pairs [bp]) indicating the presence of R. oryzae, and 15 (18.29%) showed only a single band (175bp), suggesting the presence of non-R. oryzae Mucorales. DNAs from 10 microscopically negative samples and 4 samples with septate hyphae in microscopy were successfully amplified in PCR. Considering Calcofluor white fluorescence microscopy as the gold standard for laboratory diagnosis of mucormycosis, the duplex PCR assay utilized in this study exhibited a sensitivity of 93.88%, a specificity of 100%, a negative predictive value of 91.18%, and a positive predictive value of 100% for detecting mucormycosis in IFRS specimens. The duplex PCR assay demonstrated higher sensitivity compared to direct examination with KOH (82 vs. 73) and culture (82 vs. 41), enabling rapid detection/identification of Mucorales even in samples with negative culture or in biopsies with only a few hyphal elements.

Acinetobacter baumannii Carbapenemase Producers in Morocco: Genetic Diversity.

Carbapenem resistance in Acinetobacter baumannii(A. baumannii) is a public health problem worldwide. Although carbapenem resistance is emerging in Morocco, few studies have shown the epidemiological profile of carbapenemase genes in Moroccan healthcare facilities. The aim of this study was to characterize the molecular profile of the carbapenemase enzyme in Acinetobacter baumannii from clinical isolates. Clinical strains isolated in the laboratory from various samples were subjected to several phenotypic tests. Antibiotic susceptibility and identification were tested using Phoenix 100 (Becton Dickinson Co., Sparks, MD, USA) and Api 20 (bioMérieux,Marcy-l'Etoile,France). Simple phenotypic assays were used to detect carbapenemase oxacillinase (OXA) and metallo-β-lactamase (MBL) production, including the modified Hodge test (MHT) and ethylenediaminetetraacetic acid (EDTA) test. The detection of carbapenemase genes was performed by multiplex and simple polymerase chain reaction (PCR). A total of 140 strainsor 100% of isolatescontained OXA-51 and ISbA1 sequences, 89% contained OXA-23 and OXA-58 sequences, and 1% contained OXA-24 sequence. The MBL genes were predominated by Verona integron-encodedmetallo-β-lactamase (VIM) (56%), followed by Seoul imipenemase (SIM) (39%), German imipenemase (GIM) (37%), São Paulo metallo-β-lactamase (SPM) (13%), imipenemase (IMP) (11%), and New Delhi metallo-β-lactamase (NDM) (4%). Guyana extended-spectrum β-lactamase (GES) was not found in any isolation. Our study shows a high frequency of carbapenem resistance in Acinetobacter baumannii, as it reports a high molecular diversity of carbapenemase-encoding genes, mainly dominated by the carbapenemase ISaba1/OXA-23, which represents an emerging threat in our hospital.

A Simple, Rapid, and Cost-Effective PCR Procedure for Detection of NUDT15 Gene Variants in Vietnamese Patients with Acute Lymphoblastic Leukemia.

Objective The NUDT15 variants impact thiopurine dose selection in acute lymphoblastic leukemia patients. The ability to rapidly detect variants is important in clinical practice. This study aims to develop a simple polymerase chain reaction (PCR) procedure for detecting NUDT15 variants in Vietnamese patients. Materials and Methods Sanger sequencing was used to determine NUDT15 variants from 200 patients. We designed primers and optimized the PCR procedure for detection of wild-type and variant alleles and compared with Sanger sequencing results. Results The inserted variant c.55_56insGAGTCG was detected by differences in size through conventional PCR. The tetra-primer amplification refractory mutation system PCR was successful in detecting two variations, c.52G > A and c.415C > T. The sensitivity and specificity of PCR procedure achieved 100% when compared to 200 Sanger sequencing results. Conclusion Our PCR procedure is suitable for replacing Sanger sequencing to detect the NUDT15 variants in clinical setting.

Detection of infectious agents in equine pregnancy loss, stillbirth and neonatal death

Equine abortion, stillbirth and neonatal death cause major economic losses to the equine industry worldwide. Both non-infectious and a wide range of infectious causes have been described. However, the relative contribution of pathogens to equine abortion, stillbirth and neonatal death is poorly documented, since available studies involve only a limited number of pathogens. Therefore, the objectives of the present retrospective monitoring study were to determine the prevalence of infectious agents associated with equine abortion and perinatal mortality in Belgium, and to set up a protocol usable under field conditions using polymerase chain reaction targeting. A real-time simple polymerase chain reaction for eight different abortifacient pathogens was conducted leading to the detection of at least one infectious agent in 37% of 105 analyzed cases. In the diagnosed cases, equine herpesvirus-1 was the most detected pathogen (49%), followed by Streptococcus equi subspecies zooepidemicus (28%), Coxiella burnetii (18%), Leptospira interrogans (3%) and Neospora caninum (3%). None of the analyzed cases was positive for equine viral arteritis, equine herpesvirus-4 and Chlamydophila spp. In this study, PCR targeting is shown to have its value to detect a (co)-infectious cause in equine abortion, stillbirth and neonatal death, especially in field conditions where autolysis and contamination might preclude a full post-mortem examination protocol based on classical microbiological examination.

더덕 속 식물의 구분을 위한 InDel 마커의 개발

Background: Three medicinal plants of the Campanulaceae, Codonopsis lanceolata, C. pilosula, and C. ussuriensis are perennial vines of the genus Codonopsis, and are widely disturbuted in East Asia. C. lanceolata, C. pilosula, and C. ussuriensis have been used as medicine to alleviate inflammation, increase red blood cell count and hemoglobin levels, and improve spleen and stomach functions, respectively. The three species have similar morphologies, although their pharmacological effects differ.BRMethods and Results: We identified polymorphic loci by comparing chloroplast and mitochondrial sequences of C. lanceolata, C. pilosula, and C. ussuriensis. We designed primers based on these polymorphic loci and conducted polymerase chain reaction (PCR) analysis to verify the polymorphisms. Three insertion–deletion (InDel) markers were ultimately developed: CD-mt-02, which identified C. pilosula; CD-mt-06, which identified C. ussuriensis; and CD-cp-07, which identified all the three species.BRConclusions: The three InDel markers developed in this study easily distinguished the three plant species based on a simple PCR test.

A simple PCR-SSP method for detection of HLA-B*15:02, *15:13, and *15:21

Abstract Objectives This study aims to develop a simple polymerase chain reaction (PCR) sequence-specific primer method, which will be used to genotype HLA-B*15:02, HLA-B*15:13, and HLA-B*15:21. Methods DNA was extracted from whole blood using a commercial DNA extraction kit. New specific primers were designed. The PCR was optimized for reproducibility and specificity. Parameters investigated included concentrations of MgCl2, primers concentration, and annealing temperature, to produce specific bands of interest. Known DNA samples were selected at random and tested using the PCR method. Results Simultaneously six duplex PCRs were successfully optimized using 1.0 mM MgCl2 and an annealing temperature of 62 °C. The method was also reproducible and specific. The results from the method showed 100% concordance with known DNA samples. Conclusions This study has successfully developed a simple and specific method to simultaneously genotype HLA-B*15:02, HLA-B*15:13, and HLA-B*15:21 using in-house developed PCR-SSP.

A conserved active site PenA β-lactamase Ambler motif specific for Burkholderia pseudomallei/B. mallei is likely responsible for intrinsic amoxicillin-clavulanic acid sensitivity and facilitates a simple diagnostic PCR assay for melioidosis

Burkholderia pseudomallei is a soil- and water-dwelling Gram-negative bacterium that causes melioidosis in humans and animals. Amoxicillin-clavulanic acid (AMC) susceptibility has been hailed as an integral part of the screening algorithm for identification of B. pseudomallei, but the molecular basis for the inherent AMC susceptibility of this bacterium remains undefined. This study showed that B. pseudomallei (and the closely-related B. mallei) wild-type strains are the only Burkholderia spp. that contain a 70STSK73 PenA Ambler motif. This motif was present in >99.5% of 1820 analysed B. pseudomallei strains and 100% of 83 analysed B. mallei strains, and is proposed as the likely cause for their inherent AMC sensitivity. The authors developed a polymerase chain reaction (PCR) assay that specifically amplifies the penA70ST(S/F)K73-containing region from B. pseudomallei and B. mallei, but not from the remaining B. pseudomallei complex species or the 70STFK73 region from the closely-related penB of B. cepacia complex species. The abundance and purity of the 193-bp PCR fragment from putative B. pseudomallei isolates from clinical and environmental samples is likely sufficient for reliable confirmation of the presence of B. pseudomallei. The PCR assay is designed to be especially suited for use in resource-constrained areas. While not further explored in this study, the assay may allow diagnosis of putative B. mallei in culture isolates from animal and human samples.

Production of CFTR Mutant Gene Model by Homologous Recombination System.

The most common mutation in cystic fibrosis (CF), (ΔF508-CFTR), results in impaired protein maturation, folding and transportation to the surface of the cell. As a consequence of impaired protein maturation and/or transport from the extracellular matrix to the cell, different systems are influenced, including gastrointestinal system and glandular system, reproductive system and respiratory systems. CF models are essential tools to provide further knowledge of CF pathophysiology. With this aim, we designed a transgenic CF model based on the homologous recombination (HR) system. In this experimental study, a specifically designed construct containing the CFTR gene with F508del was cloned into a PTZ57R cloning vector and then the construct was transformed into the male pronucleus by microinjection after in vitro fertilization (IVF). Then the rates of blastocyst formation and embryonic development at 72 hours after IVF, were evaluated using the inverted microscope and the insertion of the construct was approved by polymerase chain reaction (PCR) method. The CFTR gene was successfully cloned into the PTZ57R cloning vector and overall, from 22 injected cells, 5 blastocysts were observed after pronuclear injection of the CFTR gene construct. PCR verification of the blastocyst with CFTR-specific primers represented complete recombination of CFTR into the mouse genome. For the first time we designed a unique genome construction that can be detected using a simple PCR method. The pronuclear injection was performed for the transformation of the genome construct into the male pronuclei using microinjection and the development of zygote to the blastocyst stage has been observed following transgenesis.

Molecular Sexing of Nothobranchius furzeri Embryos and Larvae.

Differences between the sexes are of increasing interest in basic and applied research with regard to development, behavior, aging, and diseases. Although the African turquoise killifish Nothobranchius furzeri, a model for aging research well known for its remarkably short life span, develops strong sexual dimorphism in adulthood, there is no visible indicator of its sex in embryonic and juvenile stages. To address this issue, we developed a molecular sexing assay exploiting two large sequence polymorphisms in the minimal sex-determining region (SDR) of N. furzeri These polymorphisms are sequence deletions on the Y chromosome that involve the lack or truncation of one or multiple microsatellites. The simple polymerase chain reaction (PCR) readout of the assays described here allows the sexing of N. furzeri embryos and larvae in a medium- to high-throughput and cost-efficient manner.

Rapid membrane-based photothermal PCR for disease detection

The worldwide incidence of deadly infectious diseases has increased in recent years. Therefore, a rapid and accurate nucleic acid diagnostic system is in high demand. Polymerase chain reaction (PCR) is the standard method for amplifying and quantifying nucleic acid biomarkers for disease detection. Although several rapid PCR systems have been developed to overcome the limitations of conventional PCR, such as post-processing for analysis, complicated handling, and highly specialized instruments, a new PCR testing approach is still required. In this study, we developed a rapid membrane-based photothermal PCR that enables quantitative fluorescence detection. It requires no post-PCR procedures and provides user-friendly micro-scale solution control and sample loading. Au nanostructure was deposited on the reaction pad as a light-to-heat converter for PCR thermocycling. The automatically controlled 785 nm light source heated a solution in the reaction pad with photothermal effect. The temperature of the reaction solution, which was absorbed in a membrane, was cycled 25 times between 63 °C and 95 °C within 6 min with a photothermal cycler. The amplified samples on the membrane were then quantitatively analyzed by measuring the fluorescence signal of intercalating SYBR dye within 12 min. DNA samples extracted from Staphylococcus aureus and unpurified cells were amplified and analyzed. This membrane-based PCR approach can provide easy connectivity with various membrane-based sample pre-treatment processes, and therefore the proposed procedure could be used as the basis for an all-in-one molecular diagnosis platform. This simple, rapid, and sensitive PCR device could also serve as the basis for advanced molecular diagnostics.

Simultaneous Detection of Four Main Foodborne Pathogens in Ready-to-Eat Food by Using a Simple and Rapid Multiplex PCR (mPCR) Assay.

The increasing consumption of organic or ready-to-eat food may cause serious foodborne disease outbreaks. Developing microbiological culture for detection of food-borne pathogens is time-consuming, expensive, and laborious. Thus, alternative methods such as polymerase chain reaction (PCR) are usually employed for outbreaks investigation. In this work, we aimed to develop a rapid and simple protocol for the simultaneous detection of Escherichia coli (E coli), Listeria monocytogenes (L. monocytogenes), Staphylococcus aureus (S. aureus) and Salmonella enterica (S. enterica), by the combination of an enrichment step in a single culture broth and a multiplex PCR (mPCR) assay. The effectiveness of several enrichment media was assessed by culture and PCR. Buffered peptone water (BPW) was selected as the optimum one. Then, mPCR conditions were optimized and applied both to pure co-cultures and artificially inoculated food samples (organic lettuce and minced meat). In the culture medium inoculated at 100 CFU/mL, mPCR was able to detect the four microorganisms. When performed on artificially food samples, the mPCR assy was able to detect E. coli, S. enterica, and L. monocytogenes. In conclusion, BPW broth can effectively support the simultaneous growth of E. coli, S. aureus, L. monocytogenes, and S. enterica and could be, thus, used prior to a mPCR detection assay in ready-to-eat food, thereby considerably reducing the time, efforts and costs of analyzes.

PCR-based assay to detect the EPSPS TAP-IVS substitution in Amaranthus hybridus

Background Amaranthus spp. are problematic weeds and competitors for nutrients in several crops, especially in soybean and corn. Resistance to glyphosate has been detected in several weed species, and a triple mutation in its EPSPS target gene was detected recently in Amaranthus hybridus. Objective The aim of this work was to develop a simple polymerase chain reaction (PCR) method to detect the EPSPS triple mutation in A. hybridus. Methods Two pairs of primers were designed for PCR-based detection of [...]

BRAF Mutations in Papillary Thyroid Carcinoma: A Genomic Approach Using Probe-Based DNA Capture for Next-Generation Sequencing.

TheBRAF V600Emutation in papillary thyroid carcinoma is the major mutation in classical subtype of papillary thyroid carcinoma and other cancers.Itis the most studied predictor of clinical and pathological characteristics as well as molecular targets for cancer therapy. On the other hand, there is potential for many more forms of activating mutation in BRAF that are not detectable by simple assays to detect V600E, or even simple polymerase chain reaction (PCR)-based sequencing for full-length BRAF. Such activating mutations could arise from larger-scale rearrangements which may apparently leave no sequence change to BRAF while causing increased expression or activation by unusual means, such as gene fusion. Detection of these kinds of changes can take place using a variety of methods, though capture-based sequencing can identify the existence of such forms of mutant BRAF without needing foreknowledge of the loci involved in these kinds of mutation. In this chapter, we detail a method for capture of specific DNA sequences and their amplification to prepare for massively parallel sequencing.

DNA diagnostics for reliable and universal identification of Helicobacter pylori.

Reliable diagnostics are a major challenge for the detection and treatment of Helicobacter pylori (H. pylori) infection. Currently at the forefront are non-invasive urea breath test (UBT) and stool antigen test (SAT). Polymerase chain reaction (PCR) is not endorsed due to nonspecific primers and the threat of false-positives. The specificity of DNA amplification can be achieved by nested PCR (NPCR), which involves two rounds of PCR. If the primers are properly designed for the variable regions of the 16S rRNA gene, it is not difficult to develop an NPCR assay for the unambiguous identification of H. pylori. Elaborate NPCR for a 454 bp amplicon was validated on 81 clinical biopsy, stool, and saliva samples, each from the same individuals, and compared with available H. pylori assays, namely histology, rapid urease test, SAT, and 13C-UBT. The assay was much more sensitive than simple PCR, and it was equally sensitive in biopsy samples as the 13C-UBT test, which is considered the gold standard. In addition, it is sufficiently specific because sequencing of the PCR products exclusively confirmed the presence of H. pylori-specific DNA. However, due to the threshold and lower abundance, the sensitivity was much lower in amplifications from stool or saliva. Reliable detection in saliva also complicates the ability of H. pylori to survive in the oral cavity aside from and independent of the stomach. The reason for the lower sensitivity in stool is DNA degradation; therefore, a new NPCR assay was developed to obtain a shorter 148 bp 16S rRNA amplicon. The assay was validated on stool samples from 208 gastroenterological patients and compared to SAT results. Surprisingly, this NPCR revealed the presence of H. pylori in twice the number of samples as SAT, indicating that many patients are misdiagnosed, not treated by antibiotics, and their problems are interpreted as chronic. Thus, it is unclear how to properly diagnose H. pylori in practice. In the first approach, SAT or UBT is sufficient. If samples are negative, the 148 bp amplicon NPCR assay should be performed. If problems persist, patients should not be considered negative, but due to threshold H. pylori abundance, they should be periodically tested. The advantage of NPCR over UBT is that it can be used universally, including questionable samples taken from patients with achlorhydria, receiving proton pump inhibitors, antibiotics, bismuth compound, intestinal metaplasia, or gastric ulcer bleeding.

Development of a Quantitative One-Step RT-PCR Method for the Detection of Sabin 2 Virus Contamination in a Novel Oral Poliovirus Vaccine Type 2.

To control circulating vaccine-derived type 2 poliovirus outbreaks, a more genetically stable novel Oral Poliovirus Vaccine type 2 (nOPV2) was developed by targeted modifications of Sabin 2 genome. Since the use of OPV2 made of Sabin 2 strain has been stopped, it is important to exclude the possibility that batches of nOPV2 are contaminated with Sabin 2 virus. Here, we report the development of a simple quantitative one-step reverse-transcription polymerase chain reaction assay for the detection and quantitation of Sabin 2 virus in the presence of overwhelming amounts of nOPV2 strain. The method is specific and linear within 8 log10 range even in the presence of relevant amounts of nOPV2 virus. It is sensitive, with a lower limit of detection of 0.2 CCID50/mL (an equivalent of 198 genome copies per mL), and generates reproducible results. This assay can be used for quality control and lot release of the nOPV2.