Loop-mediated isothermal amplification of single pollen grains.

The polymerase chain reaction (PCR) has been a reliable and fruitful method for many applications in ecology. Nevertheless, unavoidable technical and instrumental requirements of PCR have limited its widespread application in field situations. The recent development of isothermal DNA amplification methods provides an alternative to PCR, which circumvents key limitations of PCR for direct amplification in the field. Being able to analyze DNA in the pollen cloud of an ecosystem would provide very useful ecological information, yet would require a field-enabled, high-throughput method for this potential to be realized. Here, we demonstrate the applicability of the loop-mediated DNA amplification method (LAMP), an isothermal DNA amplification technique, to be used in pollen analysis. We demonstrate that LAMP can provide a reliable method to identify species from the pollen cloud, and that it can amplify successfully with sensitivity down to single pollen grains, thus opening the possibility of field-based, high-throughput analysis.

Generating a Sustained Oxygen-Stable Atomic Concentration in a High-Temperature Gas Effect Investigation.

Modulated laser absorption spectroscopy is an ideal technique for evaluating flow-field parameters and determining flow-field quality by measuring the atoms dissociated in high-temperature environments. However, to obtain the absolute number density of atoms in the flow field, it is necessary to compare the measured modulated absorption spectroscopy signal with a known atomic concentration and establish a quantitative relationship through concentration calibration. Nevertheless, it remains a challenging task to prepare transient atomic samples with known concentrations that meet the calibration requirements. This study utilized the alternating-current glow discharge technique to dissociate oxygen in the air flow, resulting in the continuous generation of oxygen atoms. The absolute number densities of the generated oxygen atoms were determined by measuring the direct absorption spectra of centered on 777 nm for oxygen atoms. The number densities of the generated atoms were finely tuned by adjusting the discharge parameters. Throughout the 120-min continuous operation of the discharge system, the concentration of excited-state oxygen atoms remained stable within the range of (2.51 ± 0.02) × 108 cm-3, demonstrating the remarkable stability of the transient atomic concentration generated by the glow discharge plasma. This observation suggests that the generated atoms can be utilized as a standardized atomic sample of known concentration for absolute concentration calibration purposes.

A LAMP Assay for the Detection of Thecodiplosis japonensis, an Alien Gall Midge Species Pest of Pine Trees.

Pine needle gall midge (T. japonensis), native to Japan, has become a serious invasive pest in South Korea and, more recently in 2006, in China. It was first discovered in Qingdao, Shandong Province, and has caused serious damage to local Pinus thunbergii. The insect's small size makes morphological-based identification difficult; therefore, molecular detection techniques are urgently needed for monitoring and preventing its further spread. At present, there is no simple and accurate field molecular identification tool. To solve this problem, a LAMP-based molecular diagnosis technology of T. japonensis was developed. Four LAMP primers were designed to specifically amplify T. japonensis DNA. Positive LAMP reactions usually produce amplification in one hour. The optimal incubation conditions for LAMP detection were determined with 4 LAMP primers for 60 min at 61 °C. The LAMP detection range of gDNA concentrations is wide, with a minimum detectable gDNA concentration of 300 fg. A non-destructive DNA-releasing procedure, HotSHOT "HS6", which could extract "crude DNA" for LAMP assay in 10 min, was used for larval and adult samples. Therefore, we established a LAMP-based rapid molecular identification method that can be applied in the monitoring and management of T. japonensis.

A LAMP (loop-mediated isothermal amplification) test for rapid identification of Khapra beetle (Trogoderma granarium).

BACKGROUND: Khapra beetle (Trogoderma granarium Everts) is a significant pest of food products around the world, causing great losses of stored grain and produce, with export restrictions imposed on countries with established beetle populations. Khapra beetle is a high-priority exotic invertebrate pest in many countries requiring a rapid quarantine/biosecurity response when incursions occur. To address this, we developed a novel Khapra LAMP (loop-mediated isothermal amplification) assay using a portable real-time fluorometer and an additional 18S ribosomal DNA (18S) insect control LAMP assay for confirmation of the presence of insect DNA. Both LAMP tests can be performed either in a portable real-time fluorometer or using simple, visual colorimetric technique. RESULTS: Both the Khapra and 18S LAMP tests amplify positive samples within ≤ 25 min, with an anneal derivative temperature of 77.7 ± 0.7 °C for Khapra LAMP test and 88.0 ± 1.0 °C for 18S. The new Khapra LAMP assay is sensitive to very low levels of DNA (1.02 × 10-6  ng µL-1 ). Additionally, we developed a gBlock double stranded DNA fragment for use as positive Khapra control with a different anneal derivative of 80 °C. Both assays are simple to use in the field and are capable of amplifying DNA from target beetles, even when samples are partially degraded which is typically found during surveillance activities. By screening a broad panel of Dermestidae species we demonstrate that our new assay is species-specific, with no detections of false positives. Also, we evaluated multiple DNA extraction methods, with both QuickExtract and HotSHOT extraction methods proving suitable for in-field use. CONCLUSION: The novel Khapra and 18S LAMP assays should improve speed, accuracy and confidence of detection of Khapra beetle at incursion points and aid rapid biosecurity responses in any country affected, especially as the assays described here are portable and easy to implement in the field conditions where resources are limited. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

DNA Extraction from Plant Leaves Using a Microneedle Patch.

Isolation of high-quality DNA from infected plant specimens is an essential step for the molecular detection of plant pathogens. However, DNA isolation from plant cells surrounded by rigid polysaccharide cell walls involves complicated steps and requires benchtop laboratory equipment. As a result, plant DNA extraction is currently confined to well-equipped laboratories and sample preparation has become one of the major hurdles for on-site molecular detection of plant pathogens. To overcome this hurdle, a simple DNA extraction method from plant leaf tissues has been developed. A microneedle (MN) patch made of polyvinyl alcohol (PVA) can isolate plant or pathogenic DNA from different plant species within a minute. During DNA extraction, the polymeric MN patch penetrates into plant leaf tissues and breaks rigid plant cell walls to isolate intracellular DNA. The extracted DNA is polymerase chain reaction (PCR) amplifiable without additional purification. This minimally invasive method has successfully extracted Phytophthora infestans DNA from infected tomato leaves. Moreover, the MN patch could be used to isolate DNA from other plant pathogens directly in the field. Thus, it has great potential to become a rapid, on-site sample preparation technique for plant pathogen detection. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Microneedle patch-based DNA extraction Support Protocol 1: Microneedle patch fabrication Support Protocol 2: Real-time PCR amplification of microneedle patch extracted DNA.

Development of a bead-agglutination assay for rapid detection of Tritrichomonas foetus.

Tritrichomonas foetus is a flagellated protozoan parasite that causes inflammation of the reproductive tract leading to early embryonic death and abortion in cattle, thereby resulting in significant economic losses. Testing and culling infected bulls is an important strategy for parasite control. Routine testing is mainly limited to bulls that are traveling across state lines or within states that have specific control programs. Both culture and PCR detection methods are available, but they are not typically conducted as part of a yearly breeding soundness program and are not easily conducted in the field. In the present study, we developed a bead agglutination assay for detection of T. foetus antigens. Our experiments revealed that latex beads conjugated to T. foetus lipophosphoglycan-binding antibodies visibly clump in the presence of T. foetus. The detection limit of the assay, determined using both field and laboratory isolates of the parasite, was 0.25µg/mL and 1.0µg/mL total T. foetus antigen, respectively. Our results indicate that an antigen detection test could offer a tool for screening bulls under field conditions.