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).

Rapid detection methods and modelling simulations provide new insights into cyanobacteria detection and bloom management in a tropical reservoir.

The increasing occurrence of cyanobacteria blooms is of global concern, and is often associated with environmental and socio-economic problems, such as degenerated ecosystems and aquaculture impairment. The diazotrophic cyanobacterium Raphidiopsis raciborskii (R. raciborskii) grows rapidly in the tropics, and produces the toxin, cylindrospermopsin (CYN), which has harmful effects on aquatic organisms. Thus, to protect water quality and ecosystem, it is essential to have rapid and reliable methods for cyanobacteria and R. raciborskii detection and prediction so that early warning can be provided for management. Molecular assays, such as PCR, real-time quantitative PCR (qPCR), two-step PCR assays are accurate and widely used, but still require several hours from sample preparation to data analysis. In this study, insulated isothermal PCR (iiPCR) assays in conjunction with fast DNA extraction method, were developed and verified as a rapid detection assay in detecting cyanobacteria and R. raciborskii within 50 min, and also with high detection accuracy (98.8%) and the overall high agreement level (98.8%, k = 97.5%)) comparing to conventional qPCR assay. However, the limitation of the iiPCR assay is that it only generates qualitative results. Therefore, the quantified iiPCR assay, named as A-iiPCR, by coupling iiPCR device with fluorescence signal catching and interpretation instrument (Andor spectrometer with Solis spectroscopy software) was developed and verified with in situ environmental samples. The fluorescence intensity decreased accordingly with the drop of DNA concentration until reaching 1.32 ng/µL. Also, Delft 3D modelling was established to simulate R. raciborskii change in predicting spatial and temporal variabilities for reservoir management, as the simulated R. raciborskii concentration was the highest at sampling site 1, as well as temporally highest in April and October, posing as the most high-risk location and time periods for R. raciborskii bloom-forming requiring corresponding governance measures.

Development of a Duplex Insulated Isothermal PCR Assay for Rapid On-Site Detection and Differentiation of Genotypes 1 and 2 of African Swine Fever Virus.

Genotype II African swine fever virus (ASFV) has been plaguing Asian pig industry since 2018. Recently, genotype I ASFV was reported for the first time in China. Since there is no commercial vaccine available against ASFV, early onsite detection and quick culling procedures are commonly used by many countries all over the world. It is important that the above two genotypes of ASFV could be quickly differentiated during onsite detection at the same time. In this study, we established a sensitive and simple Fluorescent Probe Hydrolysis-Insulated isothermal PCR (iiPCR) that can detect and differentiate two genotypes of ASFV within 40 minutes. The positive or negative results of tested samples were displayed on the screen of the device automatically after PCR amplification was complete. The detection limit of the iiPCR was tested to be 20 copies for both genotype I and genotype II ASFVs. There was no cross-reactivity with other swine viruses by using the established iiPCR. Fifty-eight ASFV positive samples confirmed by National ASF Reference Laboratory were subjected to the established duplex iiPCR for genotype differentiation. The results showed that all these ASFV-positive samples belong to genotype II. At last, we found serum samples could be directly used as the templates for iiPCR without comprising sensitivity and specificity. Therefore, the duplex iiPCR established in study provide a useful tool for ASFV onsite detection and genotype differentiation.

Validation of a point-of-need diagnostic tool for rapid diagnosis of norovirus gastroenteritis.

BACKGROUND: The genogroups GI and GII of norovirus (NoV) ribonucleic acid (RNA) genetic variants are the most prevalent cause of acute gastroenteritis outbreaks, especially in children, worldwide. A fast, accurate and convenient tool for diagnosis of NoV may be preferable to the more complicated performance of real-time reverse transcription-polymerase chain reaction (RT-PCR). METHODS: In this study, we developed and evaluated a tool using insulated isothermal PCR (iiPCR)-mediated POCKIT Central NoV GI and NoV GII assay systems for diagnosis of NoV infection in pediatric patients suspected with gastroenteritis. RESULTS: Performance of POCKIT Central Norovirus GI and GII assays using RT-iiPCR, compared to regular real-time RT-PCR showed the same diagnosis rate to NoV GI (100% of total percent agreement and 1.0 of Cohen's kappa value) and a similar detection rate to norovirus GII (96.3% of total percent agreement and 0.92 of Cohen's kappa value). In exclusivity tests, the POCKIT Central NoV GI and GII assays showed negative results to other viruses, indicating that the assays may be a NoV-specific detection tool. CONCLUSION: POCKIT Central NoV GI and GII Assay systems can provide a simple, rapid, sensitive, and specific point-of-need diagnostic tool for the detection of NoV GI and GII RNAs in clinical specimens from children with acute gastroenteritis.

A Field-Deployable Insulated Isothermal PCR (iiPCR) for the Global Surveillance of Toxoplasma gondii Infection in Cetaceans.

Toxoplasmosis is a zoonotic disease with veterinary and public health importance worldwide. Toxoplasma gondii infection in cetaceans is an indicator of land-to-sea oocyst pollution. However, there is a critical knowledge gap within the distribution of the T. gondii infection in cetaceans. To facilitate the global surveillance of this important zoonotic pathogen, we developed a field-deployable duplex insulated isothermal PCR (iiPCR) with automated magnetic bead-based DNA extraction for the on-site detection of T. gondii in stranded cetaceans. It targets the B1 gene of T. gondii combined with ß2-microglobulin (B2M) gene of cetaceans as an internal control. Compared with the conventional qPCR assay, B1/B2M duplex iiPCR assay showed comparable sensitivity (21~86 bradyzoites in 25 mg of tissue) to detect spike-in standard of T. gondii DNA in cerebrum, cerebellum, skeletal muscle and myocardium tissues. Moreover, the overall agreement between the duplex iiPCR and qPCR was in almost perfect agreement (92%; 95% CI: 0.78-0.90; κ = 0.84) in detecting a synthetic spike-in standards. The B1/B2M iiPCR assay coupled with a field-deployable system provides a prompt (~1.5 h), feasible, highly sensitive and specific on-site diagnostic tool for T. gondii in stranded cetaceans. This platform provides one approach to evaluating aquatic ecosystem health and developing early warnings about negative impacts on humans and marine animals.

Development and validation of an insulated isothermal PCR assay for the rapid detection of Mannheimia haemolytica.

We developed a rapid insulated isothermal PCR (iiPCR) assay for on-site detection of Mannheimia haemolytica using a primer and probe set targeting the superoxide dismutase (sodA) gene. Our iiPCR assay detected M. haemolytica clinical isolates successfully and produced negative results on other bovine or ovine respiratory pathogens, including Histophilus somni, Bibersteinia trehalosi, Trueperella pyogenes, Streptococcus suis, and Mycoplasma spp., indicating that the PCR reactions were specific. Additionally, our iiPCR assay detected as few as 21 copies of genomic DNA and 17.2 cfu/mL of bacterial culture, which was 10 and 100 times more sensitive than conventional PCR, respectively. Our iiPCR assay can be performed on a portable device in a total of 58 min and may be a useful tool for the detection of M. haemolytica in bovine and ovine respiratory disease in the field.

Evaluation of insulated isothermal PCR devices for the detection of avian influenza virus.

Highly pathogenic avian influenza viruses (HPAIVs) and low pathogenic avian influenza viruses (LPAIVs) represent important threats to the poultry industry and global human health. Due to the high rates of avian influenza virus (AIV) transmission, controlling AIV outbreaks is challenging. HPAIV is known to be transmitted from wild birds to domestic ducks, from which it can be transmitted to layer and broiler chickens. Therefore, surveillance of AIV in domestic ducks and chickens in advance of outbreaks can prevent its spread and enable timely implementation of disease control measures. Certain molecular diagnostic tools can be applied in the field for faster AIV detection. In this study, we evaluated the AIV-detection ability of two insulated isothermal PCR (iiPCR) devices, POCKIT™Micro DUO Nucleic Acid Analyzer (POCKIT DUO) and POCKIT™ Central Nucleic Acid Analyzer (POCKIT Central). We found that the analytical, in vivo and clinical performances of the two POCKIT devices were comparable to those of real-time reverse transcription PCR. Due to their brief protocols and short detection times, POCKIT DUO and POCKIT Central represent promising molecular diagnostic devices for the reliable detection of AIV.

Clinical validation of an automated reverse transcription-insulated isothermal PCR assay for the detection of severe acute respiratory syndrome coronavirus 2.

To determine clinical performance of the single-target SARS-CoV-2 orf 1 ab reverse transcription-insulated isothermal PCR (RT-iiPCR) assay, the positive percentage agreement between this assay and a laboratory real-time RT-PCR assay was 96.8% (30 of 31; 95% confidence interval [CI], 90.5%-100%) and the negative percentage agreement was 97.1% (67 of 69; 95% CI, 93.1%-100%).

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. METHOD: In 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. RESULTS: All 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. CONCLUSION: This bimA iiPCR/POCKIT assay will undoubtedly complement other methodologies used in the clinical laboratory for the rapid identification of this pathogen.

Development and evaluation of a real-time RT-PCR and a field-deployable RT-insulated isothermal PCR for the detection of Seneca Valley virus.

BACKGROUND: Seneca Valley virus (SVV) has emerged in multiple countries in recent years. SVV infection can cause vesicular lesions clinically indistinguishable from those caused by other vesicular disease viruses, such as foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), vesicular stomatitis virus (VSV), and vesicular exanthema of swine virus (VESV). Sensitive and specific RT-PCR assays for the SVV detection is necessary for differential diagnosis. Real-time RT-PCR (rRT-PCR) has been used for the detection of many RNA viruses. The insulated isothermal PCR (iiPCR) on a portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, SVV rRT-PCR and RT-iiPCR were developed and validated. RESULTS: Neither the SVV rRT-PCR nor the RT-iiPCR cross-reacted with any of the vesicular disease viruses (20 FMDV, two SVDV, six VSV, and two VESV strains), classical swine fever virus (four strains), and 15 other common swine viruses. Analytical sensitivities of the SVV rRT-PCR and RT-iiPCR were determined using serial dilutions of in vitro transcribed RNA as well as viral RNA extracted from a historical SVV isolate and a contemporary SVV isolate. Diagnostic performances were further evaluated using 125 swine samples by two approaches. First, nucleic acids were extracted from the 125 samples using the MagMAX™ kit and then tested by both RT-PCR methods. One sample was negative by the rRT-PCR but positive by the RT-iiPCR, resulting in a 99.20% agreement (124/125; 95% CI: 96.59-100%, κ = 0.98). Second, the 125 samples were tested by the taco™ mini extraction/RT-iiPCR and by the MagMAX™ extraction/rRT-PCR system in parallel. Two samples were positive by the MagMAX™/rRT-PCR system but negative by the taco™ mini/RT-iiPCR system, resulting in a 98.40% agreement (123/125; 95% CI: 95.39-100%, κ = 0.97). The two samples with discrepant results had relatively high CT values. CONCLUSIONS: The SVV rRT-PCR and RT-iiPCR developed in this study are very sensitive and specific and have comparable diagnostic performances for SVV RNA detection. The SVV rRT-PCR can be adopted for SVV detection in laboratories. The SVV RT-iiPCR in a simple field-deployable system could serve as a tool to help diagnose vesicular diseases in swine at points of need.

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.

Evaluation of a field-deployable reverse transcription-insulated isothermal PCR for rapid and sensitive on-site detection of Zika virus.

BACKGROUND: The recent emergence of Zika virus (ZIKV) in Brazil and its precipitous expansion throughout the Americas has highlighted the urgent need for a rapid and reliable on-site diagnostic assay suitable for viral detection. Such point-of-need (PON), low-cost diagnostics are essential for ZIKV control in vulnerable areas with limited resources. METHODS: We developed and evaluated a ZIKV-specific field-deployable RT-iiPCR reagent set targeting the E gene for rapid detection of ZIKV in ZIKV-spiked human and mosquito specimens, and compared its performance to the Center for Disease Control and Prevention (CDC) and Pan American Health Organization (PAHO) RT-qPCR assays targeting the E and NS2B genes, respectively. RESULTS: These assays demonstrated exclusive specificity for ZIKV (African and Asian lineages), had limits of detection ranging from 10 to 100 in vitro transcribed RNA copies/µl and detection endpoints at 10 plaque forming units/ml of infectious tissue culture fluid. Analysis of human whole blood, plasma, serum, semen, urine, and mosquito pool samples spiked with ZIKV showed an agreement of 90% (k = 0.80), 92% (k = 0.82), 95% (k = 0.86), 92% (k = 0.81), 90% (k = 0.79), and 100% (k = 1), respectively, between the RT-iiPCR assay and composite results from the reference RT-qPCR assays. Overall, the concurrence between the ZIKV RT-iiPCR and the reference RT-qPCR assays was 92% (k = 0.83). CONCLUSIONS: The ZIKV RT-iiPCR has a performance comparable to the reference CDC and PAHO RT-qPCR assays but provides much faster results (~1.5 h) with a field-deployable system that can be utilized as a PON diagnostic with the potential to significantly improve the quality of the health care system in vulnerable areas.

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.

Field-Deployable Reverse Transcription-Insulated Isothermal PCR (RT-iiPCR) Assay for Rapid and Sensitive Detection of Foot-and-Mouth Disease Virus.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals, which can decimate the livestock industry and economy of countries previously free of this disease. Rapid detection of foot-and-mouth disease virus (FMDV) is critical to containing an FMD outbreak. Availability of a rapid, highly sensitive and specific, yet simple and field-deployable assay would support local decision-making during an FMDV outbreak. Here we report validation of a novel reverse transcription-insulated isothermal PCR (RT-iiPCR) assay that can be performed on a commercially available, compact and portable POCKIT™ analyser that automatically analyses data and displays '+' or '-' results. The FMDV RT-iiPCR assay targets the 3D region of the FMDV genome and was capable of detecting 9 copies of in vitro-transcribed RNA standard with 95% confidence. It accurately identified 63 FMDV strains belonging to all seven serotypes and showed no cross-reactivity with viruses causing similar clinical diseases in cloven-hoofed animals. The assay was able to identify FMDV RNA in multiple sample types including oral, nasal and lesion swabs, epithelial tissue suspensions, vesicular and oral fluid samples, even before the appearance of clinical signs. Clinical sensitivity of the assay was comparable or slightly higher than the laboratory-based real-time RT-PCR assay in use. The assay was able to detect FMDV RNA in vesicular fluid samples without nucleic acid extraction. For RNA extraction from more complex sample types, a commercially available taco™ mini transportable magnetic bead-based, automated extraction system was used. This assay provides a potentially useful field-deployable diagnostic tool for rapid detection of FMDV in an outbreak in FMD-free countries or for routine diagnostics in endemic countries with less structured laboratory systems.

Comparison of electron microscopy, ELISA, real time RT-PCR and insulated isothermal RT-PCR for the detection of Rotavirus group A (RVA) in feces of different animal species.

There is no gold standard for detection of Rotavirus Group A (RVA), one of the main causes of diarrhea in neonatal animals. Sensitive and specific real-time RT-PCR (rtRT-PCR) assays are available for RVA but require submission of the clinical samples to diagnostic laboratories. Patient-side immunoassays for RVA protein detection have shown variable results, particularly with samples from unintended species. A sensitive and specific test for detection of RVA on the farm would facilitate rapid management decisions. The insulated isothermal RT-PCR (RT-iiPCR) assay works in a portable machine to allow sensitive and specific on-site testing. The aim of this investigation was to evaluate a commercially available RT-iiPCR assay for RVA detection in feces from different animal species. This assay was compared to an in-house rtRT-PCR assay and a commercially available rtRT-PCR kit, as well as an ELISA and EM for RVA detection. All three PCR assays targeted the well-conserved NSP5 gene. Clinical fecal samples from 108 diarrheic animals (mainly cattle and horses) were tested. The percentage of positive samples by ELISA, EM, in-house rtRT-PCR, commercial rtRT-PCR, and RT-iiPCR was 29.4%, 31%, 36.7%, 51.4%, 56.9%, respectively. The agreement between different assays was high (81.3-100%) in samples containing high viral loads. The sensitivity of the RT-iiPCR assay appeared to be higher than the commercially available rtRT-PCR assay, with a limit of detection (95% confidence index) of 3-4 copies of in vitro transcribed dsRNA. In conclusion, the user-friendly, field-deployable RT-iiPCR system holds substantial promise for on-site detection of RVA.

Rapid screening of roundup ready soybean in food samples by a hand-held PCR device.

Insulated isothermal PCR (iiPCR) method was recently available for rapid on-site detection of roundup ready soybean (RRS; event GTS40-3-2) in food materials and products. Performance of this method was evaluated in this study. The 100% detection endpoint for the RRS by iiPCR was found in samples containing 0.1% RRS, equivalent to the results of the reference real-time PCR (rtPCR). Analysis of nucleic acids of soybean-based processed food products indicated 95% agreement between the iiPCR and rtPCR for RRS detection. By testing soybean milk and tofu samples using simple pretreatment methods, we found that the agreements between iiPCR and rtPCR methods of the aforementioned samples were 80% and 90%, respectively. Replicated tests of all discrepant samples implied that these samples had trace amounts of RRS, suggesting that the iiPCR system is more sensitive than the rtPCR method. In conclusion, the iiPCR technology can be a useful point-of-need tool to help make a timely decision in the consumption of genetically modified organisms.

An insulated isothermal PCR method on a field-deployable device for rapid and sensitive detection of canine parvovirus type 2 at points of need.

Canine parvovirus type 2 (CPV-2), including subtypes 2a, 2b and 2c, causes an acute enteric disease in both domestic and wild animals. Rapid and sensitive diagnosis aids effective disease management at points of need (PON). A commercially available, field-deployable and user-friendly system, designed with insulated isothermal PCR (iiPCR) technology, displays excellent sensitivity and specificity for nucleic acid detection. An iiPCR method was developed for on-site detection of all circulating CPV-2 strains. Limit of detection was determined using plasmid DNA. CPV-2a, 2b and 2c strains, a feline panleukopenia virus (FPV) strain, and nine canine pathogens were tested to evaluate assay specificity. Reaction sensitivity and performance were compared with an in-house real-time PCR using serial dilutions of a CPV-2b strain and 100 canine fecal clinical samples collected from 2010 to 2014, respectively. The 95% limit of detection of the iiPCR method was 13 copies of standard DNA and detection limits for CPV-2b DNA were equivalent for iiPCR and real-time PCR. The iiPCR reaction detected CPV-2a, 2b and 2c and FPV. Non-targeted pathogens were not detected. Test results of real-time PCR and iiPCR from 99 fecal samples agreed with each other, while one real-time PCR-positive sample tested negative by iiPCR. Therefore, excellent agreement (k = 0.98) with sensitivity of 98.41% and specificity of 100% in detecting CPV-2 in feces was found between the two methods. In conclusion, the iiPCR system has potential to serve as a useful tool for rapid and accurate PON, molecular detection of CPV-2.