Isothermal Polymerase Chain Reaction Research Articles

Development and validation of an insulated isothermal polymerase chain reaction assay for the rapid detection of Mycoplasma synoviae.

Mycoplasma synoviae, which causes the disease known as chicken synovitis, causes serious immunosuppression. We developed a rapid insulated isothermal polymerase chain reaction (iiPCR) assay for on-site detection of M. synoviae using a primer and probe set targeting the variable lipoprotein and haemagglutinin (vlhA) gene. In addition, the specificity, sensitivity, repeatability, and clinical detection of this method were evaluated. Our iiPCR assay detected M. synoviae clinical isolates and samples successfully and produced negative results on Mycoplasma galliscepticum, avian viral arthritis, Escherichia coli, Salmonella, Staphylococcus aureus and Corynebacterium, indicating that the PCR reactions were specific. Additionally, our iiPCR assay detected the prepared positive standard plasmid diluted 10 times (1.00 × 10-1 - 1.00 × 10-10) as a template. The undiluted positive plasmid was positive and double distilled water was negative indicating that the PCR reactions were sensitive, respectively. Finally, the vlhA positive standard plasmid with dilution multiple of 1.00 × 10-4 - 1.00 × 10-6 was repeatedly detected three times to evaluate the repeatability of the iiPCR method established in this experiment showing that the iiPCR of M. synoviae is repeatable. The established iiPCR was also used to detect 50 chicken joint enlargement samples. The thermostatic detection PCR established in this experiment was comparable to a reference real-time PCR (qPCR).

Nucleic acid amplification‐based methods for diagnosis of shrimp viral diseases

AbstractViral diseases are one of the constraints affecting the sustainability of the shrimp farming industry worldwide. The common causes of viral infections include white spot syndrome virus, infectious hypodermal and haematopoietic necrosis virus, hepatopancreatic parvovirus, Penaeus monodon nudivirus, decapod iridescent virus 1, Taura syndrome virus, yellow head virus, infectious myonecrosis virus and Macrobrachium rosenbergii nodavirus. Accurate diagnostic methods are necessary for preventing the spread of these transboundary diseases. Many molecular diagnostic methods for shrimp viral diseases have been developed and are currently in use, including conventional polymerase chain reaction (PCR), nested PCR, loop‐mediated isothermal amplification PCR, recombinase polymerase assay and real‐time PCR. Although the World Organization for Animal Health (founded as OIE) recommends several PCR methods for shrimp disease diagnosis, there has been a lack of efforts to comparatively evaluate the performance of the available methods, resulting in a significant knowledge gap. Therefore, in this review, we compare various PCR assays and commercial PCR‐based diagnostic kits that have been developed and/or published previously, and discuss pros and cons of each of these methods. Our objective of this review is to offer enhanced clarity and a comprehensive overview of molecular diagnostic methods for shrimp viral diseases. This endeavour would serve as an important resource for researchers engaged in relevant studies and professionals within the shrimp‐farming industry, providing valuable guidance and insights.

On-site detection system of Candidatus Liberibacter asiaticus by using TaqMan probe-based insulated isothermal polymerase chain reaction (iiPCR).

Citrus Huanglongbing (HLB) is one of the most destructive diseases of citrus plants caused by the obligate and phloem-limiting bacterium Candidatus Liberibacter asiaticus (Las). Reliable detection methods are important for successful control of the disease. This study was aimed to develop a rapid and user-friendly on-site detection system for Las using the TaqMan probe-based insulated isothermal polymerase chain reaction (iiPCR) assay. The Las-specific on-site detection system could be completed within one hour by simple DNA extraction coupled with a portable POCKIT device, which can perform PCR amplification and automatically provide qualitative results derived from fluorescence signals. The sensitivity of the TaqMan probe-iiPCR assay could be as low as single copy of Las, comparable to a real-time PCR method. Further testing of the field citrus samples showed 100% agreement between the TaqMan probe-iiPCR assay and the real-time PCR method, and the on-site detection system also demonstrated a great performance of Las detection. With high specificity and sensitivity, the on-site detection system developed in this study becomes a simple, rapid and powerful tool for detecting Las in fields.

Rapid Detection of Avian Infectious Bronchitis Virus by Using Commercial Insulated Isothermal Polymerase Chain Reaction (iiPCR) and Enzyme-Linked Immunosorbent Assay (ELISA)

The Journal of Agro-Technology and Rural Sciences is published by the University of Colombo Institute for Agro-Technology and Rural Sciences, Hambantota, Sri Lanka. It provides an academic pathway for professionals and researchers to contribute ground-breaking work in the field of agricultural technology and rural sciences including crop science, animal science, soil science, agricultural biology, food science and technology, postharvest technology, agricultural engineering, agricultural economics and extension.

Rapid detection of Actinobacillus pleuropneumoniae targeting the apxIVA gene for diagnosis of contagious porcine pleuropneumonia in pigs by polymerase spiral reaction.

Actinobacillus pleuropneumoniae is the primary aetiological agent of contagious porcine pleuropneumonia associated with serious economic impact on pig husbandry worldwide. Diagnosis of the disease by existing techniques including isolation and identification of bacteria followed by serotyping, serological techniques, conventional PCR, real-time PCR and LAMP assays are cumbersome, time-consuming, costly and not suitable for rapid field application. A novel isothermal polymerase chain reaction (PSR) technique is standardized for all the reagents, incubation time and incubation temperature against A. pleuropneumoniae. The sensitivity of the assay was determined against various dilutions of purified DNA and total bacterial count. The specificity of the assay was determined against 11 closely related bacterial isolates. The relative sensitivity and specificity were compared with bacterial isolation, conventional PCR and real-time PCR assays. The PSR assay for specific detection was standardized at 64°C for 30 min of incubation in a water bath. The result was visible by the naked eye after centrifugation of the reaction mixture or after incorporation of SYBR Green dye as yellowish-green fluorescence. The technique was found to be 100% specific and equally sensitive with real-time PCR and 10 times more sensitive than conventional PCR. The PSR assay could be applicable in the detection of the organisms in porcine nasal swabs spiked with A. pleuropneumoniae. This is the first-ever report on the development of PSR for specific detection of A. pleuropneumoniae and can be applied for early diagnosis at the field level.

Evaluation of a New Norovirus Genogroups GI and GII In Vitro Molecular Diagnostic Assay Using Clinical Specimens Collected from Acute Diarrheal Outbreaks.

Norovirus is a leading cause of acute gastroenteritis (AGE) in Taiwan. To improve diagnosis as part of laboratory surveillance, AGE surveillance was conducted using a new fluorescent probe hydrolysis-based insulated isothermal polymerase chain reaction (PCR) method, the POCKIT system, and the results were compared with those obtained from conventional methods. A total of 119 clinical stool samples from reported AGE outbreaks were collected for this study. From 83 real-time reverse transcription PCR (rRT-PCR) norovirus-positive cases, the POCKIT system identified 78 with a sensitivity of 90.3% in GI genogroup and 96.7% in GII genogroup. The specificity for both GI and GII genogroups was 100%. Overall, the POCKIT system is faster and easier to use than the conventional rRT-PCR method, and because of its high sensitivity and specificity, this system is a promising alternative for the detection of norovirus in patients with AGE, and would benefit public health laboratories for near real-time surveillance of AGE epidemic outbreaks.

The Sensitivity and Specificity of Loop-Mediated Isothermal Amplification and PCR Methods in Detection of Foodborne Microorganisms: A Systematic Review and Meta-Analysis.

Background:The loop-mediated isothermal amplification (LAMP) method is frequently used for identifying many microorganisms. The present review aimed to evaluate the sensitivity and specificity of LAMP method for detection of food-borne bacteria and to compare these features with those of polymerase chain reaction (PCR), as an alternative molecular diagnostic procedure, and with cultivation method, as the gold standard method.Methods:The literature was searched in electronic databases (PubMed, Scopus, Web of Science, and EMBASE) for recruiting publications within Jan 2000 to Jul 2021. We used the combinations of keywords including foodborne disease, LAMP, PCR, Loop-mediated isothermal amplification, and polymerase chain reaction. Meta-analysis was used to adjust the correlation and heterogeneity between the studies. The efficiency of the methods was presented by negative likelihood ratio, positive likelihood ratio, sensitivity, specificity, and odds ratio using forest plots. A P-value less than 0.05 was considered as statistical significance cut off. The confidence intervals were presented at the 95% interval.Results:Overall, 23 relevant studies were analyzed. The sensitivities of LAMP and PCR methods were estimated to be 96.6% (95% CI: 95.0–97.7) and 95.6% (95%CI: 91.5–97.8), respectively. The specificities of LAMP and PCR were also estimated to be 97.6% (95%CI: 92.6–99.3) and 98.7% (95%CI: 96.5–99.5), respectively.Conclusion:The specificities of LAMP and PCR assays were determined by comparing their results with cultivation method as the gold standard. Overall, the specificity of both PCR and LAMP methods was low for detection of fastidious bacteria. Nevertheless, LAMP and PCR methods have acceptable specificities and sensitivities, and their application in clinical practice necessitates more studies.

Performance of rapid diagnostic tests, microscopy, loop-mediated isothermal amplification (LAMP) and PCR for malaria diagnosis in Ethiopia: a systematic review and meta-analysis

BackgroundRapid accurate diagnosis followed by effective treatment is very important for malaria control. Light microscopy remains the “golden standard” method for malaria diagnosis. Diagnostic test method must have sufficient level of accuracy for detecting malaria parasites. Therefore, this study aimed to investigate the diagnostic accuracy of rapid diagnostic tests (RDTs), microscopy, loop-mediated isothermal amplification (LAMP) and/or polymerase chain reaction (PCR) for the malaria diagnosis in Ethiopia.MethodsData bases such as PubMed, PubMed central, Science direct databases, Google scholar, and Scopus were searched from September to October, 2020 for studies assessing the diagnostic accuracy of RDTs, microscopy, LAMP and PCR methods for malaria diagnosis.ResultsA total of 29 studies published between 2001 and 2020 were analysed using review manager, Midas (Stata) and Meta-disc. The sensitivity and specificity of studies comparing RDT with microscopy varies from 79%–100% to 80%–100%, respectively. The sensitivity of LAMP (731 tests) was 100% and its specificity was varies from 85 to 99% when compared with microscopy and PCR. Considerable heterogeneity was observed between studies included in this meta-analysis. Meta-regression showed that blinding status and target antigens were the major sources of heterogeneity (P < 0.05). RDT had an excellent diagnostic accuracy (Area under the ROC Curve = 0.99) when compared with microscopy. Its specificity was quite good (93%–100%) except for one outlier (28%), but lower “sensitivity” was observed when PCR is a reference test. This indicates RDT had a good diagnostic accuracy (AUC = 0.83). Microscopy showed a very good diagnostic accuracy when compared with PCR.ConclusionsThe present study showed that microscopy and RDTs had high efficiency for diagnosing febrile malaria patients. The diagnostic accuracy of RDT was excellent when compared with microscopy. This indicates RDTs have acceptable sensitivities and specificities to be used in resource poor settings as an alternative for microscopy. In this study, LAMP showed an excellent sensitivities and specificities. Furthermore, the need of minimum equipment and relatively short time for obtaining results can made LAMP one of the best alternatives especially for accurate diagnosis of asymptomatic malaria.

Evaluation of an automated insulated isothermal polymerase chain reaction system for rapid and reliable, on-site detection of African swine fever virus.

To evaluate the utility of an automated insulated isothermal PCR (iiPCR) system for rapid and reliable on-site detection of African swine fever virus (ASFV) in swine biological samples. Lymph node, tissue homogenate, whole blood, serum, spleen, and tonsil samples collected from swine in North and South Vietnam. Analytic sensitivity of the iiPCR system was determined by serial dilution and analysis of 2 samples (swine tissue homogenate and blood) predetermined to be positive for ASFV. Analytic specificity was assessed by analysis of 2 samples predetermined to be negative for ASFV and positive or negative for other swine pathogens (classical swine fever virus, porcine reproductive and respiratory syndrome virus, foot-and-mouth disease virus, and porcine circovirus type 2). Diagnostic performance of the iiPCR system for detection of ASFV was determined by analysis of the various tissue sample types. For all tests, a real-time PCR assay was used as the reference method. The iiPCR system was able to detect ASFV in swine blood or tissue homogenate at dilutions up to 106, whereas the real-time PCR assay was able to detect dilutions of up to 105 or 106. The iiPCR system had high analytic specificity for detection of ASFV versus other swine pathogens. Between 97% and 100% agreement was found between results of the iiPCR system for the various tissue samples and results of real-time PCR assay. The evaluated iiPCR system was found to be a rapid, reliable, and sample-flexible method for ASFV detection and may be useful for disease surveillance and quarantine in national strategies for early ASF control.

Isothermal Nucleic Acid Amplification Technologies for the Detection of Equine Viral Pathogens

Simple SummaryEquine viral diseases remain a prominent concern for human and equine health globally. Many of these viruses are of primary biosecurity concern to countries that import equines where these viruses are not present. In addition, several equine viruses are zoonotic, which can have a significant impact on human health. Current diagnostic techniques are both time consuming and laboratory-based. The ability to accurately detect diseases will lead to better management, treatment strategies, and health outcomes. This review outlines the current modern isothermal techniques for diagnostics, such as loop-mediated isothermal amplification and insulated isothermal polymerase chain reaction, and their application as point-of-care diagnostics for the equine industry.The global equine industry provides significant economic contributions worldwide, producing approximately USD $300 billion annually. However, with the continuous national and international movement and importation of horses, there is an ongoing threat of a viral outbreak causing large epidemics and subsequent significant economic losses. Additionally, horses serve as a host for several zoonotic diseases that could cause significant human health problems. The ability to rapidly diagnose equine viral diseases early could lead to better management, treatment, and biosecurity strategies. Current serological and molecular methods cannot be field-deployable and are not suitable for resource-poor laboratories due to the requirement of expensive equipment and trained personnel. Recently, isothermal nucleic acid amplification technologies, such as loop-mediated isothermal amplification (LAMP) and insulated isothermal polymerase chain reaction (iiPCR), have been developed to be utilized in-field, and provide rapid results within an hour. We will review current isothermal diagnostic techniques available to diagnose equine viruses of biosecurity and zoonotic concern and provide insight into their potential for in-field deployment.

Laboratory evaluation of two point-of-care detection systems for early and accurate detection of influenza viruses in the Lao People’s Democratic Republic

BackgroundWe evaluated molecular-based point-of-care influenza virus detection systems in a laboratory prior to a field evaluation of on-site specimen testing. MethodsThe performance characteristics of 1) insulated isothermal polymerase chain reaction (PCR) on a POCKIT™ device and 2) real-time reverse transcription-PCR (rRT-PCR) on a MyGo Mini™ device were evaluated using human clinical specimens, beta-propiolactone-inactivated influenza viruses, and RNA controls. The rRT-PCR carried out on a CXF-96™ real-time detection system was used as a gold standard for comparison. ResultsBoth systems demonstrated 100% sensitivity and specificity and test results were in 100% agreement with the gold standard. POCKIT™ only correctly identified influenza A (M gene) in clinical specimens due to the unavailability of typing and subtyping reagents for human influenza viruses, while MyGo Mini™ had either a one log higher or the same sensitivity in detecting influenza viruses in clinical specimens compared to the gold standard. For inactivated viruses and/or viral RNA, the analytic sensitivity of POCKIT™ was shown to be comparable to, or more sensitive, than the gold standard. The analytic sensitivity of MyGo Mini™ had mixed results depending on the types and subtypes of influenza viruses. ConclusionsThe performance of the two systems in a laboratory is promising and supports further evaluation in field settings.

Rapid identification of melioidosis agent by an insulated isothermal PCR on a field-deployable device.

Background Burkholderia pseudomallei causes melioidosis, a serious illness that can be fatal if untreated or misdiagnosed. Culture from clinical specimens remains the gold standard but has low diagnostic sensitivity.MethodIn this study, we developed a rapid, sensitive and specific insulated isothermal Polymerase Chain Reaction (iiPCR) targeting bimA gene (Burkholderia Intracellular Motility A; BPSS1492) for the identification of B. pseudomallei. A pair of novel primers: BimA(F) and BimA(R) together with a probe were designed and 121 clinical B. pseudomallei strains obtained from numerous clinical sources and 10 ATCC non-targeted strains were tested with iiPCR and qPCR in parallel.ResultsAll 121 B. pseudomallei isolates were positive for qPCR while 118 isolates were positive for iiPCR, demonstrating satisfactory agreement (97.71%; 95% CI [93.45–99.53%]; k = 0.87). Sensitivity of the bimA iiPCR/POCKIT assay was 97.52% with the lower detection limit of 14 ng/µL of B. pseudomallei DNA. The developed iiPCR assay did not cross-react with 10 types of non-targeted strains, indicating good specificity.ConclusionThis bimA iiPCR/POCKIT assay will undoubtedly complement other methodologies used in the clinical laboratory for the rapid identification of this pathogen.

Biodiversity of mosquitoes and Mansonia uniformis as a potential vector of Wuchereria bancrofti in Hulu Sungai Utara District, South Kalimantan, Indonesia.

Background and Aim:Lymphatic filariasis, also known as elephantiasis, still remains a problem in Indonesia. The primary causative species of this disease are the filarial worms Wuchereria bancrofti and Brugia spp. This study was conducted to identify the diversity of species and behavior of mosquitoes and to determine the mosquitoes that could be potential vectors of filariasis.Materials and Methods:Mosquito samples derived from Hulu Sungai Utara (HSU) district in the 2017 multicenter study conducted in Indonesia were used in this cross-sectional study. The diversity of mosquito species was analyzed using the Shannon–Wiener diversity index. Mosquitoes were identified based on their species, and their DNA was isolated by polymerase chain reaction (PCR). Transcription-insulated isothermal PCR method was used to detect microfilariae/filaria larvae in the mosquitoes.Results:Biodiversity was found in 14 species of mosquitoes belonging to five genera. The maximum number of mosquitoes was recorded from the species Mansonia dives, Culex vishnui, Culex quinquefasciatus, and Mansonia uniformis. W. bancrofti infection was detected in M. uniformis at an infectivity rate of 0.3% (n=311).Conclusion:To the best of our knowledge, this is the first report of M. uniformis species as a vector of W. bancrofti in HSU district, Indonesia. More efficient and accurate studies are required to aid in the lymphatic filariasis elimination programs in this subregion.

Rapid and sensitive detection of Staphylococcus aureus in processed foods using a field‐deployed device to perform an insulated isothermal polymerase chain reaction‐based assay

AbstractThis investigation we evaluated the feasibility of using the insulated isothermal polymerase chain reaction (iiPCR) reagent in a POCKIT system to detect Staphylococcus aureus in foods by comparing its results with those obtained using quantitative PCR (qPCR). The iiPCR/POCKIT system could specifically detect S. aureus and Staphylococcus spp. and differentiate them from 10 other foodborne pathogens. In a sensitivity analysis, the iiPCR/POCKIT system had a 100% (3/3) hit rate in detecting 102–105 CFU/mL S. aureus that had been spiked in skimmed milk. Three food samples that were spiked with 0.25 CFU/mL S. aureus—skimmed milk, vegetable mixture, and cooked chicken—yielded positive results following overnight enrichment. Finally, 24 food samples that were bought from traditional markets or convenience stores were tested. The iiPCR/POCKIT system and qPCR yielded the same results for 23 of the samples, excluding one papaya milk sample, indicating that the iiPCR/POCKIT system can be an effective way for the sensitive and automatic detection of S. aureus in various processed foods.Practical applicationsStaphylococccus aureus is a major foodborne pathogen worldwide. Traditional methods for detecting S. aureus are time consuming and laborious, so they are difficult to perform in limited‐resource place. The iiPCR/POCKIT system is a field‐deployed device which combines magnetic bead‐based nucleic acid extraction and insulated isothermal polymerase chain reaction (iiPCR) to automatically perform qualitative detection of a specific DNA fragment. In this study, the assay performance of the fully automatic iiPCR/POCKIT system for detecting S. aureus in foods was compared with that of the reference qPCR. The iiPCR/POCKIT system exhibited good specificity by successfully differentiating 21 strains of S. aureus and two strains of Staphylococcus sp. from 12 other foodborne pathogens. After overnight enrichment, the detection limit of the iiPCR/POCKIT system was determined to be 0.25 CFU/mL in skimmed milk, vegetable mixture, and cooked chicken. In testing with 24 commercial food products, the results of the iiPCR/POCKIT system were comparable to those of qPCR and the traditional plate‐counting method. Therefore, the iiPCR/POCKIT system is suited to the routine identification of S. aureus in food industry.

Evaluation of a Field-Deployable Insulated Isothermal Polymerase Chain Reaction Nucleic Acid Analyzer for Influenza A Virus Detection at Swine Exhibitions

Rapid detection of influenza A virus (IAV) at swine exhibitions, where zoonotic transmission has occurred, can allow exhibition officials to quickly implement mitigation strategies and reduce public health risk. While laboratory diagnostic methods using PCR exist, pen-side detection of IAV can reduce lag time between sample collection and results. Portable insulated isothermal PCR (RT-iiPCR) has been used for point-of-care pathogen detection in veterinary medicine. This study compared laboratory methods of real-time reverse transcription PCR (rRT-PCR) to RT-iiPCR to determine the potential effectiveness of RT-iiPCR for detection of IAV in swine in the field. Two methods of extraction (magnetic bead and spin-column) and the two PCR platforms were used in a crossover study design to detect IAV in nasal wipes of 150 individual swine from one exhibition. Magnetic bead extraction is considered the laboratory gold standard while spin-column purification is considered the field-deployable method. IAV RNA was detected in 17 samples using Mag/rRT-PCR (reference assay) and 16 samples using Mag/RT-iiPCR (Sensitivity-S 76.5%), whereas only 14 samples using Spin/rRT-PCR (S 88.2%) and 12 samples using Spin/RT-iiPCR (field method) (S 58.8%) were positive, demonstrating a reduction in detection of viral RNA using column purification. There is moderate agreement (Cohen's kappa = 0.6575) between Mag/rRT-PCR and Spin/RT-iiPCR. There is good agreement between both PCR assays when using the same method of extraction (Mag: Cohen's kappa = 0.8203, Spin: Cohen's kappa = 0.7642). RT-iiPCR requires testing of 10 more samples than the rRT-PCR to detect disease at the 95% confidence level in a population of 300 animals with a disease prevalence of 20%. In conclusion, although there is some reduction in sensitivity, RT-iiPCR used in conjunction with spin-column purification is an acceptable method of IAV in swine detection at exhibitions where it may help reduce lag time and allow for rapid control of an IAV outbreak.

On-site detection of bovine leukemia virus by a field-deployable automatic nucleic extraction plus insulated isothermal polymerase chain reaction system

Bovine leukemia virus (BLV) is a contagious, oncogenic deltaretrovirus of cattle with a worldwide distribution. In the US, over 40% of dairy cows are infected with the virus, and evidence of its economic impact is growing. This study evaluated the performance of a field-deployable automatic nucleic acid-extraction/insulated isothermal PCR (iiPCR) system for on-site BLV-proviral DNA detection in dairy cows compared with a conventional laboratory real-time PCR (rt-PCR). Assay performance was verified in parallel tests of 36 archived blood samples with 100% agreement (κ = 1.0; n = 36) between the iiPCR and conventional rt-PCR systems, and the limit of detection of the iiPCR assay was estimated to be 4 copies (genome equivalent) per reaction. The field-deployable iiPCR system was subsequently used on-farm to test freshly collected blood samples, and showed 100% agreement (κ = 1.0; n = 32) with the laboratory rt-PCR system. Fresh blood samples were collected on a second farm and tested on both systems, also with 100% agreement (κ = 1.0; n = 34). The field-deployable iiPCR/POCKIT™ combo system performs as well as a conventional laboratory-based rt-PCR system for detection of BLV proviral DNA in whole blood and may be a useful tool for on-farm evaluation of BLV-infection status in dairy cattle.

Rapid and sensitive detection of Mycoplasma synoviae by an insulated isothermal polymerase chain reaction-based assay on a field-deployable device

Mycoplasma synoviae (MS), causing respiratory diseases, arthritis, and eggshell apex abnormalities in avian species, is an important pathogen in the poultry industry. Implementation of a biosecurity plan is important in MS infection management. Working on a field-deployable POCKIT™ device, an insulated isothermal polymerase chain reaction (iiPCR) assay has a potential for timely MS detection on the farm. The MS iiPCR assay had limit of detection 95% of about 9 genome equivalents by testing serial dilutions of a standard DNA. The detection endpoint of the assay for detection of MS genomic DNA was comparable to a reference real-time PCR. The assay did not crossreact with other important avian pathogens, including avian reovirus, Mycoplasma gallisepticum, Staphylococcus aureus, Escherichia coli, Pasteurella multocida, and Salmonella Pullorum. When 92 synovial fluid and respiratory tract swab samples collected from chickens, turkeys, and geese suspected of MS infection were tested, the clinical performance of the MS iiPCR had 97.8% agreement (Cohen's kappa value, 0.95) with that of the reference real-time PCR. In conclusion, the MS iiPCR/POCKIT™ system, working with field-deployable manual or automatic nucleic acid extraction methods, has potential to serve as a rapid and sensitive on-site tool to facilitate timely detection of MS.

Translation of a laboratory-validated equine herpesvirus-1 specific real-time PCR assay into an insulated isothermal polymerase chain reaction (iiPCR) assay for point-of-need diagnosis using POCKIT™ nucleic acid analyzer

Equine herpesvirus myeloencephalopathy (EHM), a major problem for the equine industry in the United States, is caused by equine herpesvirus-1 (EHV-1). In addition, EHV-1 is associated with upper respiratory disease, abortion, and chorioretinal lesions in horses. Here we describe the development and evaluation of an inexpensive, user-friendly insulated isothermal PCR (iiPCR) method targeting open reading 30 (ORF30) to detect both neuropathogenic and non-neuropathogenic strains on the field-deployable POCKIT™ device for point-of-need detection of EHV-1. The analytical sensitivity of the EHV-1 iiPCR assay was 13 genome equivalents per reaction. The assay did not cross react with ten non-target equine viral pathogens. Performance of the EHV-1 iiPCR assay was compared to two previously described real-time PCR (qPCR) assays in two laboratories by using 104 archived clinical samples. All 53 qPCR-positive and 46 of the 51 qPCR-negative samples tested positive and negative, respectively, by the iiPCR. The agreement between the two assays was 95.19% (confidence interval 90.48–99.90%) with a kappa value of 0.90. In conclusion, the newly developed EHV-1 iiPCR assay is robust to provide specificity and sensitivity comparable to qPCR assays for the detection of EHV-1 nucleic acid in clinical specimens.

Development of a TaqMan Probe-Based Insulated Isothermal Polymerase Chain Reaction (iiPCR) Assay for Detection of Fusarium oxysporum f. sp. cubense Race 4.

This study developed a novel and inexpensive detection method based on a TaqMan probe-based insulated isothermal polymerase chain reaction (iiPCR) method for the rapid detection of Panama disease caused by Fusarium oxysporum f. sp. cubense (Foc) race 4, which is currently among the most serious fungal vascular diseases worldwide. By using the portable POCKIT™ device with the novel primer set iiFoc-1/iiFoc-2, the Foc race 4 iiPCR assay (including DNA amplification and signal monitoring) could be completed within one hour. The developed Foc race 4 iiPCR assay is thus a user-friendly and efficient platform designed specifically for the detection of Foc race 4. The detection limit of this optimized Foc iiPCR system was estimated to be 1 copy of the target standard DNA as well as 1 fg of the Foc genomic DNA. This approach can serve as a rapid detection method for in planta detection of Foc race 4 in field-infected banana. It was concluded that this molecular detection procedure based on iiPCR has good potential for use as an efficient detection method.

Development and evaluation of a reverse transcription-insulated isothermal polymerase chain reaction (RT-iiPCR) assay for detection of equine arteritis virus in equine semen and tissue samples using the POCKIT™ system

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory and reproductive disease of horses. Most importantly, EAV induces abortion in pregnant mares and can establish persistent infection in up to 10–70% of the infected stallions, which will continue to shed the virus in their semen. The objective of this study was to develop and evaluate a reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) for the detection of EAV in semen and tissue samples. The newly developed assay had a limit of detection of 10 RNA copies and a 10-fold higher sensitivity than a previously described real-time RT-PCR (RT-qPCR). Evaluation of 125 semen samples revealed a sensitivity and specificity of 98.46% and 100.00%, respectively for the RT-qPCR assay, and 100.00% and 98.33%, respectively for the RT-iiPCR assay. Both assays had the same accuracy (99.2%, k=0.98) compared to virus isolation. Corresponding values derived from testing various tissue samples (n=122) collected from aborted fetuses, foals, and EAV carrier stallions are as follows: relative sensitivity, specificity, and accuracy of 88.14%, 96.83%, and 92.62% (k=0.85), respectively for the RT-qPCR assay, and 98.31%, 92.06%, and 95.08% (k=0.90), respectively for the RT-iiPCR assay. These results indicate that RT-iiPCR is a sensitive, specific, and a robust test enabling detection of EAV in semen and tissue samples with very considerable accuracy. Even though the RT-qPCR assay showed a sensitivity and specificity equal to virus isolation for semen samples, its diagnostic performance was somewhat limited for tissue samples. Thus, this new RT-iiPCR could be considered as an alternative tool in the implementation of EAV control and prevention strategies.