Evaluation of the effect of outer primer structure, and inner primer linker sequences, in the performance of Loop-mediated isothermal amplification.

Loop-mediated isothermal amplification, or LAMP, is nowadays the most popular isothermal nucleic acid amplification technique. This technique implements a minimum of four primers, named outer (F3/B3) and inner primers (FIP/BIP). The inner primers hybridize in two distinct regions, and some studies have reported that the usage of a linker, typically composed of four thymines, in the middle of these primers can improve assay performance. In addition to this, dual-priming oligonucleotides, DPO, have been reported to provide highly specific reducing non-specific amplifications. Considering the large number of primers implemented in LAMP assays, in the current study the suitability of DPO primers replacing regular outer primers; and their combination with different linker sequences in the inner primers were explored. The results demonstrated that replacing standard F3/B3 by DPO primers does not significantly affect that overall performance of the assay, and provides additional stability to temperature changes. This observations were consistent regardless the type of linker implemented in the inner primers, out of which in the current study a linker composed of thymines significantly outperformed the other options tested, most likely due to a combination of sequence and physical structure.

A multiplex nanoparticle-assisted polymerase chain reaction assay for detecting three canine epidemic viruses using a dual priming oligonucleotide system.

A rapid and accurate diagnosis of mixed viral infections is important for providing timely therapeutic interventions. The aim of this study was to develop a highly sensitive and specific method for the simultaneous detection of canine distemper virus (CDV), canine parvovirus (CPV) and canine coronavirus (CCV) in mixed infections by combining the high specificity of a dual priming oligonucleotide (DPO) primer system with the high sensitivity of a nanoparticle-assisted PCR (nanoPCR) assay. Under the optimised assay conditions, the multiplex DPO-nanoPCR assay developed using DPO primers was 100-fold more sensitive than the multiplex PCR assay using conventional primers. The detection limits of the multiplex DPO-nanoPCR assay for the recombinant plasmids containing the cloned CDV, CPV and CCV target sequences were 5.4 × 102, 6.5 × 102 and 1.6 × 102 copies in a 25 µL assay, respectively. No cross-reaction with other canine viruses was observed. This is the first reported use of a multiplex nanoPCR assay with the DPO primer system for the simultaneous detection of CDV, CPV and CCV in mixed infections. The high sensitivity and specificity of the assay indicated its potential for use in clinical diagnosis and field surveillance of animal epidemics.

Use of dual priming oligonucleotide system-based multiplex RT-PCR assay to detect five diarrhea viruses in pig herds in South China.

In this study, a specific and simple method based on the dual priming oligonucleotide (DPO) system was developed to simultaneously detect transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine rotavirus A (PRV-A), porcine delta coronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV), associated with the major enteric RNA viruses in pigs. The DPO system-based multiplex RT-PCR method simplified the primer design and did not require optimization of the annealing temperature. Specificity analysis revealed that the method could specifically detect TGEV, PEDV, PRV-A, PDCoV, and SADS-CoV without any cross-amplification of other circulating swine viruses. The limit of detection of the method was as low as 103-104 copies/µL plasmid of each virus. The method also had good repeatability, and obvious results were seen in three repeat experiments with an interval of 45 days. This optimized multiplex RT-PCR method was used to evaluate 181 clinical swine samples that were collected from four provinces of China between September 2016 and August 2018. The results showed that the positive detection rates of PEDV, PDCoV, SADS-CoV, PRV-A, and TGEV were 30.94% (56/181), 17.67% (32/181), 11.6% (21/181), 9.39% (17/181), and 0.55% (1/181), respectively. Mixed infection of two or more viruses was also common. The DPO system-based multiplex RT-PCR could be a useful tool for detecting enteric virus infections. This method has the advantages of easy operation, low cost, high detection efficiency, and short running time for early diagnosis in clinical cases.

Development of dual priming oligonucleotide-polymerase chain reaction (DPO-PCR) for detection of wheat component in foods.

This research is aimed to establish a DPO-PCR method for rapid detection of wheat component in foods. A pair of highly specific DPO primers was designed using the wheat housekeeping gene GAG56D as the target gene. By optimizing the PCR reaction system, the DPO-PCR detection method for wheat component in food was established, and the specificity and sensitivity of the method were determined. The established DPO-PCR method was highly specific, and there was no cross-reactivity to 22 control samples. The sensitivity was high, and the minimum detection limit was 1 ng/uL. Tests on commercially available product samples showed that the method can effectively detect wheat component of foods. The DPO-PCR method established in this study is simple and accurate, and provides an effective detection method for wheat component in foods.

Dual priming oligonucleotide (DPO)-based real-time RT-PCR assay for accurate differentiation of four major viruses causing porcine viral diarrhea.

Currently in China, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), and porcine deltacoronavirus (PDCoV) are the major causes of porcine viral diarrhea, and mixed infections in clinics are common, resulting in significant economic losses in pig industry. Here, a dual priming oligonucleotide (DPO)-based multiplex real-time SYBR Green RT-PCR assay were developed for accurately differentiating PEDV, TGEV, PoRV, and PDCoV in clinical specimens targeting the N gene of TGEV, PEDV, and PDCoV, and the VP7 gene of PoRV. Results showed that the DPO primer allowed a wider annealing temperature range (40-65 °C) and had a higher priming specificity compared to conventional primer, in which more than 3 nucleotides in the 3'- or 5'-segment of DPO primer mismatched with DNA template, PCR amplification efficiency would decrease substantially or extension would not proceed. DPO-based multiplex real-time RT-PCR method had analytical detection limit of 8.63 × 102 copies/µL, 1.92 × 102 copies/µL, 1.74 × 102 copies/µL, and 1.76 × 102 copies/µL for PEDV, TGEV, PoRV, and PDCoV in clinical specimens, respectively. A total of 672 clinical specimens of piglets with diarrheal symptoms were collected in Northeastern China from 2017 to 2018 followed by analysis using the assay, and epidemiological investigation results showed that PEDV, TGEV, PoRV, and PDCoV prevalence was 19.05%, 5.21%, 4.32%, and 3.87%, respectively. The assay developed in this study showed higher detection accuracy than conventional RT-PCR method, suggesting a useful tool for the accurate differentiation of the four major viruses causing porcine viral diarrhea in practice.

Simultaneous Detection of Bovine Rotavirus, Bovine Parvovirus, and Bovine Viral Diarrhea Virus Using a Gold Nanoparticle-Assisted PCR Assay With a Dual-Priming Oligonucleotide System.

Bovine rotavirus (BRV), bovine parvovirus (BPV), and bovine viral diarrhea virus (BVDV) are the pathogens that cause diarrhea primarily in newborn calves. A mixed infection of BRV, BPV, and BVDV makes clinical diagnosis difficult. In this study, we designed dual-priming oligonucleotide (DPO) primers the VP6 gene of BRV, VP2 gene of BPV, and 5'UTR gene of BVDV and synthesized gold nanoparticles (GNPs) with an average diameter of 10 nm. We combined the DPOs with the GNPs to develop a DPO-nanoPCR assay for detecting BRV, BPV, and BVDV. The annealing temperature, primer concentration, and GNP concentration were optimized for this assay. Compared to a conventional PCR assay, the DPO-nanoPCR assay allowed the use of a wider range of annealing temperatures (41-65°C) to effectively amplify target genes. PCR amplification was the most efficient at 56.2°C using conventional primers. The optimal volume of all the primers (10 µM) was 1.0 µL. The optimal volume of GNPs (10 nM) for all the reactions was 0.5 µL. The detection limits of DPO-nanoPCR for pMD19-T-VP6, pMD19-T-VP2, and pMD19-T-5'UTR were 9.40 × 102 copies/µL, 5.14 × 103 copies/µL, and 4.09 × 101 copies/µL, respectively; and those using conventional PCR were 9.40 × 104 copies/µL, 5.14 × 105 copies/µL, and 4.09 × 104 copies/µL, respectively. The sensitivity of DPO-nanoPCR was at least 100-fold higher than that of conventional PCR. The specificity detection showed that the DPO-nanoPCR was able to specifically detect BRV, BPV, and BVDV. Use of clinical samples indicated that target viruses can be detected accurately. Thus, DPO-nanoPCR is a new powerful, simple, specific, and sensitive tool for detecting mixed infections of BRV, BPV, and BVDV.

Cost-effectiveness of a tailored Helicobacter pylori eradication strategy based on the presence of a 23S ribosomal RNA point mutation that causes clarithromycin resistance in Korean patients.

BACKGROUND AND AIM: The Helicobacter pylori eradication rate using conventional triple therapy has decreased due to clarithromycin (CAM) resistance in H. pylori. Recently, dual priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) can be used to detect H. pylori and point mutations in the 23S ribosomal RNA gene causing CAM resistance. This study aimed to evaluate the success rate and cost-effectiveness of tailored H. pylori eradication using DPO-PCR. METHODS: The H. pylori-positive patients diagnosed by a rapid urease test or DPO-PCR were enrolled from a single academic hospital. The patients with positive rapid urease test results received a CAM-based triple regimen. In the tailored therapy group that underwent DPO-PCR testing, patients with A2142G and/or A2143G point mutations were treated with a bismuth-containing quadruple regimen. The cost-effectiveness of H. pylori eradication success was evaluated according to the average cost per patient and the incremental cost-effectiveness ratio. RESULTS: A total of 243 patients were allocated to the triple therapy group and 124 patients to the tailored therapy group. The first-line eradication rate of H. pylori was significantly higher in the tailored therapy group than in the conventional triple therapy group (92.7% vs 76.5%, P < 0.001). The average costs per patient for tailored therapy were $307.37 and $299.59 for first-line and second-line treatments, respectively. Compared with triple therapy, the incremental cost-effectiveness ratios of tailored therapy were $3.96 and -$3.81 per patient for first-line and second-line treatments, respectively. CONCLUSION: In Korea, tailored H. pylori eradication using DPO-PCR may be more cost-effective than conventional triple therapy.

Dual-priming oligonucleotide-based multiplex PCR using tissue samples from the rapid urease test kit for the detection of Helicobacter pylori in bleeding peptic ulcers.

BACKGROUND: In patients with peptic ulcer bleeding (PUB), diagnostic tests for Helicobacter pylori (H. pylori) infection have low sensitivity. The aim of our study was to investigate the diagnostic yield of dual-priming oligonucleotide-based multiplex (DPO)-PCR using tissue samples from the rapid urease test (RUT, CLO(®)test) kit in patients with PUB. METHODS: We prospectively enrolled patients with PUB. During second-look endoscopy, gastric biopsy specimens for histology and RUT were obtained from a total of 170 patients. DPO-PCR tests were performed on tissue samples obtained from the CLO(®)test kit. If testing for H. pylori was negative, endoscopy with re-biopsy was performed 8 weeks after the bleeding episode. RESULTS: H. pylori-associated bleeding was confirmed in 64.1% (109/170) of the patients. At the bleeding episode, the diagnostic sensitivities of RUT, histology, and DPO-PCR test were 47.7% (52/109), 71.6% (78/109) and 97.2% (106/109), respectively (p<0.01). The specificity of the DPO-PCR test was 91.8% (56/61). The positive predictive value (PPV) of the DPO-PCR test was 95.5% (106/111), and its negative predictive value (NPV) was 94.9% (56/59). CONCLUSIONS: In patients with PUB, the DPO-PCR test could be a useful diagnostic tool for H. pylori infection. Particularly given a negative RUT result, subsequent DPO-PCR testing of tissue samples from the CLO(®)test kit could be of considerable benefit.

Design and testing of multiplex RT-PCR primers for the rapid detection of influenza A virus genomic segments: Application to equine influenza virus.

The avian influenza A virus causes respiratory infections in animal species. It can undergo genomic recombination with newly obtained genetic material through an interspecies transmission. However, the process is an unpredictable event, making it difficult to predict the emergence of a new pandemic virus and distinguish its origin, especially when the virus is the result of multiple infections. Therefore, identifying a novel influenza is entirely dependent on sequencing its whole genome. Occasionally, however, it can be time-consuming, costly, and labor-intensive when sequencing many influenza viruses. To compensate for the difficulty, we developed a rapid, cost-effective, and simple multiplex RT-PCR to identify the viral genomic segments. As an example to evaluate its performance, H3N8 equine influenza virus (EIV) was studied for the purpose. In developing this protocol to amplify the EIV eight-segments, a series of processes, including phylogenetic analysis based on different influenza hosts, in silico analyses to estimate primer specificity, coverage, and variation scores, and investigation of host-specific amino acids, were progressively conducted to reduce or eliminate the negative factors that might affect PCR amplification. Selectively, EIV specific primers were synthesized with dual priming oligonucleotides (DPO) system to increase primer specificity. As a result, 16 primer pairs were selected to screen the dominantly circulating H3N8 EIV 8 genome segments: PA (3), PB2 (1), PA (3), NP (3), NA8 (2), HA3 (1), NS (1), and M (2). The diagnostic performance of the primers was evaluated with eight sets composing of four segment combinations using viral samples from various influenza hosts. The PCR results suggest that the multiplex RT-PCR has a wide range of applications in detection and diagnosis of newly emerging EIVs. Further, the proposed procedures of designing multiplex primers are expected to be used for detecting other animal influenza A viruses.

Molecular Approaches to Identify Helicobacter pylori Antimicrobial Resistance.

Antimicrobial susceptibility testing is needed to adapt Helicobacter pylori treatment to obtain the best results. Beside the standard phenotypic methods, molecular methods are increasingly used. The value of these molecular tests is that they are quick, independent of the transport conditions, easy to standardize, and commercial kits are available. In this article, these methods are reviewed, focusing on the determination of H pylori resistance to macrolides and fluoroquinolones, and mentioning also the methods used for tetracycline and rifampin.

Dual-priming oligonucleotide-based multiplex PCR using tissue samples in rapid urease test in the detection of Helicobacter pylori infection.

AIM: To investigate whether tissue samples processed by the rapid urease test (RUT) kit are suitable for dual-priming oligonucleotide-based multiplex polymerase chain reaction (DPO-PCR) to detect Helicobacter pylori (H. pylori). METHODS: A total of 54 patients with specific gastrointestinal symptom were enrolled in this study. During endoscopy, gastric biopsy specimens were taken for histology, RUT, and DPO-PCR. DPO-PCR was performed on gastric biopsy samples and tissue samples that were analyzed by RUT at 2 separate institutes. In detecting H. pylori, the concordance rate of the DPO-PCR tests between the tissue samples that had been submitted to RUT and the gastric biopsy samples was investigated. RESULTS: H. pylori co-occurred with 76.0% (19/25) of gastric ulcers, 64.3% (9/14) of duodenal ulcers, and 33.3% (4/12) of gastritis cases. H. pylori infection was found in 100% (3/3) of the patients with both gastric and duodenal ulcers. Overall, H. pylori was detected in 35 of 54 (64.8%) patients. The diagnostic sensitivities of histology, RUT, and DPO-PCR were 85.7% (30/35), 74.3% (26/35), and 97.1% (34/35), respectively (P = 0.02). The positive predictive value (PPV) of DPO-PCR was 94.4%, whereas the negative predictive value (NPV) was 94.7%. In the rapid urease test (CLOtest)-negative cases, the frequency of positive DPO-PCR and histologic results was 20.0% (7/35). The concordance rate of the DPO-PCR tests between the tissue samples from the RUT kit and the gastric biopsy samples was 94.4% (51/54). The rate of DPO-PCR and silver stain positivity in the RUT-negative cases was 20.0% (7/35). CONCLUSION: In diagnosing H. pylori infection, DPO-PCR can be performed on tissue samples that have been processed by the RUT kit. Particularly, in patients with RUT-negative results, DPO-PCR on these tissue samples could be helpful in detecting of H. pylori infection.

Comparison of Three BRAF Mutation Tests in Formalin-Fixed Paraffin Embedded Clinical Samples.

BACKGROUND: Recently, BRAF inhibitors showed dramatic treatment outcomes in BRAF V600 mutant melanoma. Therefore, the accuracy of BRAF mutation test is critical. METHODS: BRAF mutations were tested by dual-priming oligonucleotide-polymerase chain reaction (DPO-PCR), direct sequencing and subsequently retested with a real-time PCR assay, cobas 4800 V600 mutation test. In total, 64 tumors including 34 malignant melanomas and 16 papillary thyroid carcinomas were analyzed. DNA was extracted from formalin-fixed paraffin embedded tissue samples and the results of cobas test were directly compared with those of DPO-PCR and direct sequencing. RESULTS: BRAF mutations were found in 23 of 64 (35.9%) tumors. There was 9.4% discordance among 3 methods. Out of 6 discordant cases, 4 cases were melanomas; 3 cases were BRAF V600E detected only by cobas test, but were not detected by DPO-PCR and direct sequencing. One melanoma patient with BRAF mutation detected only by cobas test has been on vemurafenib treatment for 6 months and showed a dramatic response to vemurafenib. DPO-PCR failed to detect V600K mutation in one case identified by both direct sequencing and cobas test. CONCLUSIONS: In direct comparison of the currently available DPO-PCR, direct sequencing and real-time cobas test for BRAF mutation, real-time PCR assay is the most sensitive method.

Comparison of Three BRAF Mutation Tests in Formalin-Fixed Paraffin Embedded Clinical Samples

BACKGROUND: Recently, BRAF inhibitors showed dramatic treatment outcomes in BRAF V600 mutant melanoma. Therefore, the accuracy of BRAF mutation test is critical. METHODS: BRAF mutations were tested by dual-priming oligonucleotide-polymerase chain reaction (DPO-PCR), direct sequencing and subsequently retested with a real-time PCR assay, cobas 4800 V600 mutation test. In total, 64 tumors including 34 malignant melanomas and 16 papillary thyroid carcinomas were analyzed. DNA was extracted from formalin-fixed paraffin embedded tissue samples and the results of cobas test were directly compared with those of DPO-PCR and direct sequencing. RESULTS: BRAF mutations were found in 23 of 64 (35.9%) tumors. There was 9.4% discordance among 3 methods. Out of 6 discordant cases, 4 cases were melanomas; 3 cases were BRAF V600E detected only by cobas test, but were not detected by DPO-PCR and direct sequencing. One melanoma patient with BRAF mutation detected only by cobas test has been on vemurafenib treatment for 6 months and showed a dramatic response to vemurafenib. DPO-PCR failed to detect V600K mutation in one case identified by both direct sequencing and cobas test. CONCLUSIONS: In direct comparison of the currently available DPO-PCR, direct sequencing and real-time cobas test for BRAF mutation, real-time PCR assay is the most sensitive method.

Evaluation of the Seeplex® Meningitis ACE Detection kit for the detection of 12 common bacterial and viral pathogens of acute meningitis.

BACKGROUND: Bacterial meningitis is an infectious disease with high rates of mortality and high frequency of severe sequelae. Early identification of causative bacterial and viral pathogens is important for prompt and proper treatment of meningitis and for prevention of life-threatening clinical outcomes. In the present study, we evaluated the value of the Seeplex Meningitis ACE Detection kit (Seegene Inc., Korea), a newly developed multiplex PCR kit employing dual priming oligonucleotide methods, for diagnosing acute meningitis. METHODS: Analytical sensitivity of the kit was studied using reference strains for each pathogen targeted by the kit, while it's analytical specificity was studied using the human genome DNA and 58 clinically well-identified reference strains. For clinical validation experiment, we used 27 control cerebrospinal fluid (CSF) samples and 78 clinical CSF samples collected from patients at the time of diagnosis of acute meningitis. RESULTS: The lower detection limits ranged from 10(1) copies/µL to 5×10(1) copies/µL for the 12 viral and bacterial pathogens targeted. No cross-reaction was observed. In the validation study, high detection rate of 56.4% was obtained. None of the control samples tested positive, i.e., false-positive results were absent. CONCLUSIONS: The Seeplex Meningitis ACE Detection kit showed high sensitivity, specificity, and detection rate for the identification of pathogens in clinical CSF samples. This kit may be useful for rapid identification of important acute meningitis-causing pathogens.

Evaluation of the Seeplex(R) Meningitis ACE Detection Kit for the Detection of 12 Common Bacterial and Viral Pathogens of Acute Meningitis

BACKGROUND: Bacterial meningitis is an infectious disease with high rates of mortality and high frequency of severe sequelae. Early identification of causative bacterial and viral pathogens is important for prompt and proper treatment of meningitis and for prevention of life-threatening clinical outcomes. In the present study, we evaluated the value of the Seeplex Meningitis ACE Detection kit (Seegene Inc., Korea), a newly developed multiplex PCR kit employing dual priming oligonucleotide methods, for diagnosing acute meningitis. METHODS: Analytical sensitivity of the kit was studied using reference strains for each pathogen targeted by the kit, while it's analytical specificity was studied using the human genome DNA and 58 clinically well-identified reference strains. For clinical validation experiment, we used 27 control cerebrospinal fluid (CSF) samples and 78 clinical CSF samples collected from patients at the time of diagnosis of acute meningitis. RESULTS: The lower detection limits ranged from 101 copies/microL to 5x101 copies/microL for the 12 viral and bacterial pathogens targeted. No cross-reaction was observed. In the validation study, high detection rate of 56.4% was obtained. None of the control samples tested positive, i.e., false-positive results were absent. CONCLUSIONS: The Seeplex Meningitis ACE Detection kit showed high sensitivity, specificity, and detection rate for the identification of pathogens in clinical CSF samples. This kit may be useful for rapid identification of important acute meningitis-causing pathogens.

Diagnostic testing for Duchenne/Becker Muscular dystrophy using Dual Priming Oligonucleotide (DPO) system

PURPOSE: Large exon deletions in the DMD gene are found in about 60% of DMD/BMD patients. Multiplex PCR has been employed to detect the deletion mutation, which frequently generates noise PCR products due to the presence of multiple primers in a single reaction as well as the stringency of PCR conditions. This often leads to a false-negative or false-positive result. To address this problematic issue, we introduced the dual primer oligonucleotide (DPO) system. DPO contains two separate priming regions joined by a polydeoxyinosine linker that results in high PCR specificity even under suboptimal PCR conditions. METHODS: We tested 50 healthy male controls, 50 patients with deletion mutation as deletion-positive patient controls, and 20 patients with no deletions as deletion-negative patient controls using DPO- multiplex PCR. Both the presence and extent of deletion were verified by simplex PCR spanning the promoter region (PM) and 18 exons including exons 3, 4, 6, 8, 12, 13, 17, 19, 43-48, 50-52, and 60 in all 120 controls. RESULTS: DPO-multiplex PCR showed 100% sensitivity and specificity for the detection a deletion. However, it showed 97.1% sensitivity and 100% specificity for determining the extent of deletions. CONCLUSION: The DPO-multiplex PCR method is a useful molecular test to detect large deletions of DMD for the diagnosis of patients with DMD/BMD because it is easy to perform, fast, and cost-effective and has excellent sensitivity and specificity.

Evaluation of Multiplex PCR Assay Using Dual Priming Oligonucleotide System for Detection Mutation in the Duchenne Muscular Dystrophy Gene / 대한진단검사의학회지

BACKGROUND: Exon deletions of Duchenne muscular dystrophy (DMD) gene account for most of the alterations found in DMD and Becker muscular dystrophy (BMD). This study was to evaluate the usefulness of dual priming oligonucleotide multiplex PCR (DPO PCR) in detection of exon deletions of DMD gene. METHODS: Thirty-seven DMD or BMD patients who had known exon deletions detected by conventional multiplex PCR (conventional PCR) and nine control subjects were enrolled in this study. When a discrepancy was shown between the results of conventional PCR and DPO PCR, the multiplex ligation-dependent probe amplification (MLPA) technique was performed as a confirmation test. RESULTS: The same deletions previously identified by conventional PCR in 32 out of 37 subjects were also detected by DPO PCR. For the five subjects (13.5%) showing discrepant results between the conventional PCR and DPO PCR, MLPA was performed and its results were found to correlate better with those of DPO PCR. The discrepancies were due to false positive or false negative results of the conventional PCR. CONCLUSIONS: DPO PCR shows a high agreement of results with the conventional PCR and is considered an adequate method to be used as a primary genetic test for the diagnosis of DMD. Because of an improved accuracy, especially for determining the boundaries of DMD gene deletions, DPO PCR can be very useful as a supplement to the conventional PCR.