Current and emerging molecular technologies for the diagnosis of plant diseases – An overview

Plant diseases caused by numerous pathogens such as bacteria, viruses, and fungi are responsible for substantial economic losses in the agricultural industry worldwide. Specific, sensitive, and efficient diagnostic tools have been developed worldwide to mitigate and prevent the pathogenic threat. The diagnostic tools have revolutionized from classical methods to more advanced molecular diagnostic approaches such as enzyme-linked immunosorbent assay (ELISA), conventional polymerase chain reaction (PCR), real-time PCR, loop-mediated isothermal amplification (LAMP), biosensor, and next-generation sequencing (NGS). Hence, this review describes the current and emerging molecular diagnostic tools to distinguish and identify pathogens in crops

Biosensor DNA voltametri berasaskan nanozarah emas bersalut elektrod bercetak skrin karbon untuk pengesanan DNA organisma terubah suai genetik (GMO)

Genosensor voltametri untuk pengesanan DNA organisma terubah suai genetik (GMO) telah dibangunkan berasaskan nanozarah emas (AuNPs) dan elektrod pes karbon bercetak skrin (SPE). AuNPs telah dipegunkan pada SPE melalui kaedah pertumbuhan perantara benih dan dicirikan dengan mikroskop elektron pengimbasan (SEM). 6-Mercapto-1- heksanol (MCH) telah digunakan untuk membentuk lapisan mono prob DNA rantai tunggal (ssDNA) pada SPE terubah suai AuNPs (AuNPs-SPE). Biosensor GMO telah dioptimumkan secara elektrokimia menggunakan oligonukleotida sintetik melalui teknik voltametri denyutan pembezaan (DPV). Biosensor GMO yang dioptimumkan kemudiannya digunakan untuk menganalisis sampel sebenar bagi pengesanan khusus urutan DNA dalam virus mozek kubis bunga, iaitu gen CaMV 35S. Isyarat penghibridan DNA dipantau berdasarkan arus puncak pengoksidaan penunjuk redoks asid monosulfonat antrakuinona (AQMS) semasa interkalasinya ke dalam dupleks DNA. Biosensor GMO memberi rangsangan secara linear kepada DNA terubah suai genetik (GM) antara 0.1 nM dan 300.0 nM dan had pengesanan diperoleh pada 0.06 nM. Sisihan piawai relatif (RSD) kebolehulangan biosensor GMO dianggarkan pada 6.7-7.8%. Biosensor GMO berasaskan AuNPs mempamerkan prestasi pengesanan DNA yang lebih baik daripada segi julat linear dinamik dan had pengesanan berbanding dengan biosensor GMO elektrokimia yang dilaporkan sebelum ini. Biosensor DNA elektrokimia ini menyediakan satu platform pengesanan pakai buang untuk aplikasi dalam ujian bahan GMO

A carbon dots based fluorescence sensing for the determination of Escherichia coli O157:H7

Sensing founded on fluorescence quenching involving carbon dots (CDs) and gold nanoparticles (AuNPs) for the determination of Escherichia coli (E. coli) O157:H7 has been explored. CDs act as the fluorophore, while AuNPs as the quencher. Target oligos have been utilized to establish distance between CDs and AuNPs nanoparticles in close proximity. At excitation/emission wavelength of 340 nm/450 nm, respectively, the net CDs fluorescence quenching increased proportionally with increasing viscosity of the target oligos. A linear correlation was found between the fluorescence quenching of CDs and the logarithm concentration of target oligos in the series of 0.01–200 nM (slope = 675.6, R2 = 0.992) with the detection limit (LOD) of 1.03 ± 3.54 nM. The proposed method was utilized for verification of selectivity and specificity towards different oligonucleotide sequence and bacteria strain with satisfactory results

Real-time PCR for detection of fliC gene of E. coli O157:H7 in beef and chicken meat

The SYBR Green I real-time PCR assay was used to quantify E. coli O157:H7 in various meat samples. Primers were designed to amplify and quantify the structural flagella (fliC) gene of E. coli O157:H7 in a single reaction. The primer specificity was confirmed with DNA from an ATCC culture of E. coli O157:H7 EDL933 as positive control, autoclaved E. coli O157:H7 EDL933 as negative control (NC) and nuclease free water as non template control (NTC). A direct correlation was determined between the fluorescence threshold (Ct) and the starting quantity of E. coli O157:H7 DNA. A detection limit of 4.71 x 10–2 ng/ μl of E. coli O157:H7 DNA equivalent to approximately 1.4 x 10–2 CFU of E. coli O157:H7 ml–1 based on plate counts was determined. Quantification of E. coli O157:H7 in Australian and Malaysian beef, chicken meat, burger and minced beef from the markets was possible when DNA quantity was as low as 1.0 x 10–2 ng/μl. These results indicated that the developed PCR assay was suitable for quantitative determination of E. coli O157:H7 in meat samples

Evaluation of beak and feather disease virus, avian polyomavirus and avian papillomavirus of captives psittacine birds in Seri Kembangan, Selangor, Malaysia

Aims: Psittacine birds such as parrots, macaws, cockatoos, lovebirds and parakeets, are widely reared as household pets or at aviary due to their attractive features. However, the status of virus-causing diseases of psittacine species in Malaysia is fairly under-documented. Therefore, this study was aimed to detect the presence of three common avian viruses that infect psittacine birds, i.e. beak and feather disease virus (BFDV), avian polyomavirus and avian papillomavirus. Methodology and results: Faecal samples from twelve asymptomatic captive psittacine birds of different species were collected from an undisclosed animal garden in Serdang, Selangor, Malaysia. Briefly, the sample was homogenised and resuspended with SM buffer with the ratio 1:1 (weight of sample/g: volume of SM buffer/mL) before centrifugation at 1,000 × g for 20 min. The supernatant was collected and filtered before subjected to genomic DNA extraction using a commercialised kit. Polymerase chain reaction (PCR) technique was used to screen the V1, VP1 and L1 genes of beak and feather disease virus (BFDV), avian polyomavirus and avian papillomavirus, respectively. Findings revealed that the samples were negative for BFDV and avian polyomavirus. However, positive results of 1.5 kbp PCR amplicon were detected for avian papillomavirus in four out of the 12 samples (33.33%), which was from the white-crested cockatoo, African grey parrot, yellow-collared macaw and Senegal parrot. Sequence analysis of the L1 gene from the Senegal parrot Poicephalus senegalus revealed 93% identity to a reference Psittacus erithacus timneh avian papillomavirus. Conclusion, significance and impact of study: This study added to the limited prevalence data of three important avian viruses which infect captive psittacines in Seri Kembangan, Selangor, Malaysia. Avian papillomavirus, but not BFDV and avian polyomavirus, was detected in the collected captive psittacine birds. Therefore, a routine screening can be performed to monitor the health status of birds despite their asymptomatic manifestation, in order to prevent possible virus transmission

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Advanced DNA-Based Point-of-Care Diagnostic Methods for Plant Diseases Detection

Diagnostic technologies for the detection of plant pathogens with point-of-care capability and high multiplexing ability are an essential tool in the fight to reduce the large agricultural production losses caused by plant diseases. The main desirable characteristics for such diagnostic assays are high specificity, sensitivity, reproducibility, quickness, cost efficiency and high-throughput multiplex detection capability. This article describes and discusses various DNA-based point-of care diagnostic methods for applications in plant disease detection. Polymerase chain reaction (PCR) is the most common DNA amplification technology used for detecting various plant and animal pathogens. However, subsequent to PCR based assays, several types of nucleic acid amplification technologies have been developed to achieve higher sensitivity, rapid detection as well as suitable for field applications such as loop-mediated isothermal amplification, helicase-dependent amplification, rolling circle amplification, recombinase polymerase amplification, and molecular inversion probe. The principle behind these technologies has been thoroughly discussed in several review papers; herein we emphasize the application of these technologies to detect plant pathogens by outlining the advantages and disadvantages of each technology in detail

Molecular Inversion Probe: A New Tool for Highly Specific Detection of Plant Pathogens

<div><p>Highly specific detection methods, capable of reliably identifying plant pathogens are crucial in plant disease management strategies to reduce losses in agriculture by preventing the spread of diseases. We describe a novel molecular inversion probe (MIP) assay that can be potentially developed into a robust multiplex platform to detect and identify plant pathogens. A MIP has been designed for the plant pathogenic fungus <i>Fusarium oxysporum</i> f.sp. <i>conglutinans</i> and the proof of concept for the efficiency of this technology is provided. We demonstrate that this methodology can detect as little as 2.5 ng of pathogen DNA and is highly specific, being able to accurately differentiate <i>Fusarium oxysporum</i> f.sp. <i>conglutinans</i> from other fungal pathogens such as <i>Botrytis cinerea</i> and even pathogens of the same species such as <i>Fusarium oxysporum</i> f.sp. <i>lycopersici</i>. The MIP assay was able to detect the presence of the pathogen in infected <i>Arabidopsis thaliana</i> plants as soon as the tissues contained minimal amounts of pathogen. MIP methods are intrinsically highly multiplexable and future development of specific MIPs could lead to the establishment of a diagnostic method that could potentially screen infected plants for hundreds of pathogens in a single assay.</p></div