Evaluation of an Automatic Insulated Isothermal PCR System for Rapid and Reliable Field-Deployable Detection of African Swine Fever Virus

In the present study, we evaluate an automatic sample-to-answer insulated isothermal PCR system for rapid and reliable field-deployable detection of African swine fever virus in comparison to that of OIE recommended real-time PCR counterparts with samples collected in Vietnam. For analytical sensitivity, the system could detect ASFV up to a dilution of 106 whereas the real time PCR systems could detect up to a dilution of 105 to 106. For specificity test, the system showed high specificity to ASFV in compare to different other types of swine pathogens: PRRSV, FMDV, PCV2, CSFV. The diagnostic performance comparison on 6 different types of samples showed 97.3% to 100% agreement with reference real-time PCR. The results of this study indicated that POCKIT Central-based insulated isothermal PCR system is a rapid, reliable and sample-flexible method for effective detection of ASFV.

Development and Evaluation of an iiPCR Assay for Salmonella and Shigella Detection on a Field-Deployable PCR System

Background. Salmonella and Shigella are often associated with fecal-oral transmission and cause large-scale outbreaks in centralized catering units and, therefore, should be frequently and strictly monitored, especially among food handlers. However, no specific and sensitive on-site detection method is available until now. Methods. In this study, an insulated isothermal PCR assay for the detection of Salmonella and Shigella on a field-deployable PCR system was developed. Specificity, sensitivity, reproducibility, and clinical accuracy of the assay were characterized and evaluated. Results. The insulated isothermal PCR assay could be completed within 58 minutes with minimal pretreatment needed. The assay was specific and with good reproducibility. The limit of detection was 103 CFU/mL and 101 CFU/mL for Salmonella and Shigella, respectively, which was comparable to multiplex real-time PCR. Mock on-site clinical evaluation results showed that the analytical sensitivity and specificity of the insulated isothermal PCR assay were 100% and 96.6%, while the positive predictive value and negative predictive value were 94.1% and 100%, respectively. Conclusion. Based on our results, we believe that the assay developed herein could serve as an alternative method for preliminary screening and provide a valuable platform for the on-site detection of Salmonella and Shigella, especially in resource-limited and developing countries.

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.

Avian influenza surveillance in wild birds using sentinel ducks

We conducted active surveillance for avian influenza virus using sentinel ducks in central region of Mongolia (Khunt lake Saikhan soum, Bulgan province) that major wild bird habitat and outbreak site of H5N1 HPAI in wild birds in Mongolia from 2005 to 2011. Total of 39/104 (37,5%) samples were positive by insulated isothermal PCR (iiPCR) and 42/104 (40,38%) swab samples were positive by real time PCR (qPCR). In addition, AIV antibody detected in 35/104 (33,65%) serum samples tested by AIV NP ELISA kit. These results indicated that sentinel surveillance using domestic birds could be an effective method for avian pathogens including influenza in Mongolia. Enhanced sentinel surveillance in wild bird populations in Mongolia is therefore crucial for the understanding of global AIV transmission and epidemiology. Шувууны томуугийн тандах судалгаанд туршуул шувуу (Sentinel bird) байршуулах арга ашигласан дүнгээс Бид шувууны томуугийн тандах судалгаанд туршуул шувуу байршуулах арга зүйг ашиглах боломжийг судлах зорилгоор урьдчилсан туршилтыг 2019 оны 7-10 сард Булган аймгийн Сайхан сумын Хунт нууранд хийж гүйцэтгэв. Хунт нуур нь олон тооны нүүдлийн усны шувууд зусах болон дайрч өнгөрдөг ач холбогдолтой цэг бөгөөд 2005-2011 онд өндөр хоруу чанартай шувууны томуугийн (HPAI) A/H5N1 дэд хэвшлийн вирус илэрч байсан. Шувуунаас авсан арчдасны зарим дээжийг insulated isothermal PCR (iiPCR)-р шинжлэхэд 39/104 (37,5%), дээж эерэг, бүх дээжийг PCR (qPCR)-р шинжлэхэд 42/104 (40,38%) нь дээж эерэг дүн үзүүлсэн. Харин ийлдсэнд шувууны томуугийн эсрэг бием илрүүлэх ELISA-ийн шинжилгээгээр 35/104 (33,65%) дээжинд эсрэг бием илэрсэн. Иймд энэ арга зүйг Монгол орны нөхцөлд тохируулан сайжруулж шувууны томуугийн үүсгэгчийг илрүүлэхэд ашиглах нь уг өвчний эпидемиологийн байдлыг танин мэдэхэд чухал ач холбогдолтой юм. Түлхүүр үг: нугас, вирус, дархлаа, эпидемиологи, тархалт, ПГУ (Полимеразан гинжин урвал)

Preliminary result of insulated isothermal PCR (IIPCR) for avian influenza surveillance

In avian influenza (AI) surveillance, total 686 (fecal n=682, swabs n = 4) samples of migratory birds were collected on August 2018 in Saikhan soum, Bulgan aimag, and these samples were analyzed by portable insulated isothermal (iiPCR) PCR in the field condition. Total of 137 pool of samples (each pool contains 4-6 samples) were analyzed by iiPCR and 3 pools of sample were found positive. Total of 9 pool of samples were positive by virus isolation with embryonated egg inoculation and HA analysis and 4/9 were identified as an avian influenza and 5/9 were identified as a Paramyxovirus by RT-PCR analysis. After sequencing, H3 (n=3) and H2 (n=1) subtypes were determined. iiPCR positive 3 pools were confirmed by egg inoculation and HA analysis and it shows that this newly applied method was able to use to the surveillance of avian influenza due to usefulness and high sensitivity. Шувууны томуугийн тандах судалгаанд зөөврийн полимеразын гинжин урвалыг ашигласан дүнгээс Шувууны томуугийн тандах судалгаанд зөөврийн (insulated isothermal PCR (iiPCR)) ПГУ-ыг хээрийн нөхцөлд хэрэглэх боломжийг турших зориолгоор 2018 оны 8 сард Булган аймгийн Сайхан сум Хунт нуурны орчмоос цуглуулсан нийт 686 (сангас; n=682, арчдас; n=4) усны шувуудын дээжинд хээрийн нөхцөлд шинжилгээ хийж үр дүнг гаргав. Гарсан үр дүнг лабораторид үр хөврөлт өндгөнд өсгөвөрлөх, ЦНУ болон RT-PCR-р баталгаажуулж илэрсэн вирусын НА дэд хэвшлийг нуклеотидын дараалал тогтоох аргаар тодорхойлов. Хээрийн нөхцөлд 137 багц дээжинд (4-6 дээж = 1 багц) iiPCR-р хийсэн илрүүлэх шинжилгээгээр шувууны томуугийн вирусын генийн өвөрмөц хэсэг 3 багц дээжинд илрэв. Лабораторид вирус өсгөвөрлөх болон ЦНУ-р шинжлэхэд 9 багц дээж эерэг гарсанаас шувууны томуугийн вирус (n=4) болон шувууны парамиксовирус (n=5)-ын халдвар байгааг RT-PCR-р баталгаажуулж, гений дараалал тогтоох аргаар шувууны томуугийн H3 (n=3) болон H2 (n=1) дэд хэвшил болохыг тодорхойлов. iiPCR-аар шувууны томуугийн вирусын генийн өвөрмөц хэсэг илэрсэн 3 багц дээжийн үр дүн лабораторийн шинжилгээний дүнгээр баталгаажсан нь энэхүү хэрэглэхэд хялбар, мэдрэг чанар өндөр, түргэвчилсэн аргыг тандах судалгаанд хэрэглэх боломжтойг илтгэж байна. Түлхүүр үг: нүүдлийн шувуу, тандалт, үүсгэгч илрүүлэх, дэд хэвшил