Hepatitis A is a liver infection caused by the hepatitis A virus (HAV). Outbreaks of hepatitis A have been linked to the consumption of both raw and cooked shellfish. These outbreaks could induce a public confidence problem over shellfish safety and may result in important economic losses for the seafood industry. The work presented in this study investigated the presence of HAV in shellfish from Peninsular Malaysia. A total of 365 of cultured and wild shellfish from 36 sampling locations located throughout Peninsular Malaysia were examined using a commercial nucleic acid extraction and reverse transcription -polymerase chain reaction (RT-PCR) kit. HAV was not detected in almost all of the shellfish samples xamined. Only one cockle sample from Changkat, Seberang Perai was positive for HAV. The results suggest the absence of HAV or very low amount of HAV viral particles in most of the shellfish examined.
Foot-and-mouth disease (FMD) is a devastating livestock disease caused by foot-and-mouth disease virus (FMDV). Outbreaks of this disease in a country always result in conspicuous economic losses to livestock industry and subsequently lead to serious socioeconomic damages due to the immediate imposition of trade embargo. Rapid and accurate diagnoses are imperative to control this infectious virus. In the current review, enzyme-linked immunosorbent assay (ELISA)-based methods used in FMD diagnosis are extensively reviewed, particularly the sandwich, liquid-phase blocking, and solid-phase competition ELISA. The differentiation of infected animals from vaccinated animals using ELISA-based methods is also highlighted, in which the role of 3ABC polyprotein as a marker is reviewed intensively. Recently, more studies are focusing on the molecular diagnostic methods, which detect the viral nucleic acids based on reverse transcription-polymerase chain reaction (RT-PCR) and RT-loop-mediated isothermal amplification (RT-LAMP). These methods are generally more sensitive because of their ability to amplify a minute amount of the viral nucleic acids. In this digital era, the RT-PCR and RT-LAMP are progressing toward the mobile versions, aiming for on-site FMDV diagnosis. Apart from RT-PCR and RT-LAMP, another diagnostic assay specifically designed for on-site diagnosis is the lateral flow immunochromatographic test strips. These test strips have some distinct advantages over other diagnostic methods, whereby the assay often does not require the aid of an external device, which greatly lowers the cost per test. In addition, the on-site diagnostic test can be easily performed by untrained personnel including farmers, and the results can be obtained in a few minutes. Lastly, the use of FMDV diagnostic assays for progressive control of the disease is also discussed critically.
Nucleobases represent key structural motifs in biologically active molecules, including synthetic and natural products. Molecular modifications made on nucleobases or their isolation from natural sources are being widely investigated for the development of drugs with improved potency for the treatment of different diseases, such as cancer, as well as viral and bacterial infections. This review article focuses on the nucleobase analogue drug developments of the past 20 years (2000-2020). Various pharmacological and medicinal aspects of nucleobases and their analogues are discussed. The current state and limitations are also highlighted.
A fluorescence biosensor has been developed based on hybridisation chain reaction (HCR) amplification coupled with silver nanoclusters (AgNCs) for nucleic acid detection. The fluorescence was activated via end-to-end transfer of dark AgNCs caged within a DNA template to another DNA sequence that could enhance their red fluorescence emission at 611 nm. Such cluster-transfer approach allows us to introduce fluorogenic AgNCs as external signal transducers, thereby enabling HCR to perform in a predictable manner. The resulted HCR-AgNC biosensor was able to detect target DNA with a detection limit of 3.35 fM, and distinguish the DNA target from single-base mismatch sequences. Moreover, the bright red fluorescence emission was detectable with the naked eye, with concentration of target DNA down to 1 pM. The biosensor also performed well in human serum samples with good recovery. Overall, our cluster-transfer approach provides a good alternative to construct HCR-AgNC assay with less risk of circuit leakage and produce AgNCs in a controllable manner.
An abdominal aortic aneurysm (AAA) is abnormal swelling in the abdominal aorta and a prevalent life-threatening disease. This research introduces a new interdigitated microelectrode (IDME)-sensing surface modified by iron oxide nanoworms (IONWs) for detecting the AAA biomarker insulin-like growth factor-1 (IGF1). A sandwich pattern was formulated with the IGF1 aptamer and IGFBP1 (IGF binding protein-1) on the IONW-constructed IDME hybrid to identify IGF1. The surface morphology of the IONWs revealed a uniform distribution of worm-like structures (80-100 nm) as confirmed by FESEM and FETEM analyses. Further, the presence of the major elements, Fe and O, was confirmed by EDX and XPS studies. The crystal planes that appeared in the IONW reflect cubic magnetite. IONW-modified IDME attained a limit of detection for IGF1 of 1 fM (3σ) with an aptamer-IGF1-IGFBP1 sandwich. This sandwich with IGFBP1 enhanced the current level at all concentrations of IGF1 and displayed linearity in the range 1 fM to 100 pM with a determination coefficient of R2 = 0.9373 [y = 3.38221x - 4.79]. Control experiments with complementary aptamer sequences, IGF2 and IGFBP3 did not show notable signal changes, indicating the specific detection of IGF1. This IONW constructed electrode helps to achieve the detection of low amounts of IGF1 and diagnose AAA at the stage prior to rupture.
Antibiotic-adjuvant combinatory therapy serves as a viable treatment option in addressing antibiotic resistance in the clinical setting. This study was carried out to assess and characterize the adjuvant potential and mode of action of linalool against carbapenemase-producing Klebsiella pneumoniae (KPC-KP). Linalool exhibited bactericidal activity alone (11,250 μg/ml) and in combination with meropenem (5,625 μg/ml). Comparative proteomic analysis showed significant reduction in the number of cytoplasmic and membrane proteins, indicating membrane damage in linalool-treated KPC-KP cells. Upregulation of oxidative stress regulator proteins and downregulation of oxidative stress-sensitive proteins indicated oxidative stress. Zeta potential measurement and outer membrane permeability assay revealed that linalool increases the bacterial surface charge as well as the membrane permeability. Intracellular leakage of nucleic acid and proteins was detected upon linalool treatment. Scanning and transmission electron microscopies further revealed the breakage of bacterial membrane and loss of intracellular materials. Linalool induced oxidative stress by generating reactive oxygen species (ROS) which initiates lipid peroxidation, leading to damage of the bacterial membrane. This leads to intracellular leakage, eventually killing the KPC-KP cells. Our study demonstrated that linalool possesses great potential in future clinical applications as an adjuvant along with existing antibiotics attributed to their ability in disrupting the bacterial membrane by inducing oxidative stress. This facilitates the uptake of antibiotics into the bacterial cells, enhancing bacterial killing.
Mining of plant-derived antimicrobials is the major focus at current to counter antibiotic resistance. This study was conducted to characterize the antimicrobial activity and mode of action of linalyl anthranilate (LNA) against carbapenemase-producing Klebsiella pneumoniae (KPC-KP). LNA alone exhibited bactericidal activity at 2.5% (V/V), and in combination with meropenem (MPM) at 1.25% (V/V). Comparative proteomic analysis showed a significant reduction in the number of cytoplasmic and membrane proteins, indicating membrane damage in LNA-treated KPC-KP cells. Up-regulation of oxidative stress regulator proteins and down-regulation of oxidative stress-sensitive proteins indicated oxidative stress. Zeta potential measurement and outer membrane permeability assay revealed that LNA increases both bacterial surface charge and membrane permeability. Ethidium bromide influx/efflux assay showed increased uptake of ethidium bromide in LNA-treated cells, inferring membrane damage. Furthermore, intracellular leakage of nucleic acid and proteins was detected upon LNA treatment. Scanning and transmission electron microscopies again revealed the breakage of bacterial membrane and loss of intracellular materials. LNA was found to induce oxidative stress by generating reactive oxygen species (ROS) that initiate lipid peroxidation and damage the bacterial membrane. In conclusion, LNA generates ROS, initiates lipid peroxidation, and damages the bacterial membrane, resulting in intracellular leakage and eventually killing the KPC-KP cells.
In this work, a simple and inexpensive physical lysis method using a cordless drill fitted with a plastic pellet pestle and 150 mg of sterile sea sand was established for the extraction of DNA from six strains of freshwater microalgae. This lysis method was also tested for RNA extraction from two microalgal strains. Lysis duration between 15 and 120 s using the cetyltrimethyl ammonium bromide (CTAB) buffer significantly increased the yield of DNA from four microalgalstrains (Monoraphidium griffithii NS16, Scenedesmus sp. NS6, Scenedesmus sp. DPBC1 and Acutodesmus sp. DPBB10) compared to control. It was also found that grinding was not required to obtain DNA from two strains of microalgae (Choricystis sp. NPA14 and Chlamydomonas sp. BM3). The average DNA yield obtained using this lysis method was between 62.5 and 78.9 ng/mg for M. griffithii NS16, 42.2-247.0 ng/mg for Scenedesmus sp. NS6, 70.2-110.9 ng/mg for Scenedesmus sp. DPBC1 and 142.8-164.8 ng/mg for Acutodesmus sp. DPBB10. DNA obtained using this method was sufficiently pure for PCR amplification. Extraction of total RNA from M. griffithii NS16 and Mychonastes sp. NPD7 using this lysis method yielded high-quality RNA suitable for RT-PCR. This lysis method is simple, cheap and would enable rapid nucleic acid extraction from freshwater microalgae without requiring costly materials and equipment such as liquid nitrogen or beadbeaters, and would facilitate molecular studies on microalgae in general.
Point-of-care biosensors are important tools developed to aid medical diagnosis and testing, food safety and environmental monitoring. Paper-based biosensors, especially nucleic acid-based lateral flow assays (LFA), are affordable, simple to produce and easy to use in remote settings. However, the sensitivity of such assays to infectious diseases has always been a restrictive challenge. Here, we have successfully electrospun polycaprolactone (PCL) on nitrocellulose (NC) membrane to form a hydrophobic coating to reduce the flow rate and increase the interaction rate between the targets and gold nanoparticles-detecting probes conjugates, resulting in the binding of more complexes to the capture probes. With this approach, the sensitivity of the PCL electrospin-coated test strip has been increased by approximately ten-fold as compared to the unmodified test strip. As a proof of concept, this approach holds great potential for sensitive detection of targets at point-of-care testing.
Hepatitis A virus infection occurs globally and is causing a public health concern, primarily in developing countries due to its persistent circulation in the environment. The improved sanitary condition and increase in awareness of personal hygiene have led to the marked reduction of HAV prevalence in industrialized countries during childhood and to a shift of the infection towards adulthood. HAV is an environmentally stable, positive single stranded RNA virus that is primarily transmitted by the fecal-oral route, person to person contact or ingestion of contaminated food and drink. One of the main causes leading to HAV infection is epidemiologically linked to the consumption of raw or undercooked shellfish particularly oysters and clams. Due to their filter-feeding style, these shellfishes readily concentrate viruses from the surrounding water containing municipal sewage, and as a consequence pose a health threat to consumers. Therefore, development of detection techniques possessing the requisite sensitivity and specificity for the practical routine monitoring purposes is of great importance necessary for the protection of shellfish-consuming public. Nucleic acid based method such as reverse transcription PCR has emerged as the popular method of choice in view of its rapidity, accuracy and
sensitivity in contrary of the time-consuming conventional cell culture and hybridization techniques. However, detection of hepatitis A virus is firstly hampered by the non-cytophatic effect of wild type HAV strain, secondly, the low concentration of viral genome present in the environmental sample which requires effective isolation and concentration of virions and lastly the labor-extensive purification and thorough removal of the abundance of the PCR inhibitors which will unfavorably reduce the efficiency of PCR detection.
The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began as a cluster of pneumonia cases in Wuhan, China before spreading to over 200 countries and territories on six continents in less than six months. Despite rigorous global containment and quarantine efforts to limit the transmission of the virus, COVID-19 cases and deaths have continued to increase, leaving devastating impacts on the lives of many with far-reaching effects on the global society, economy and healthcare system. With over 43 million cases and 1.1 million deaths recorded worldwide, accurate and rapid diagnosis continues to be a cornerstone of pandemic control. In this review, we aim to present an objective overview of the latest nucleic acid-based diagnostic tests for the detection of SARS-CoV-2 that have been authorized by the Food and Drug Administration (FDA) under emergency use authorization (EUA) as of 31 October 2020. We systematically summarize and compare the principles, technologies, protocols and performance characteristics of amplification- and sequencing-based tests that have become alternatives to the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel. We highlight the notable features of the tests including authorized settings, along with the advantages and disadvantages of the tests. We conclude with a brief discussion on the current challenges and future perspectives of COVID-19 diagnostics.
Since the 1970s, people’s understanding of life has gradually deepened into the basic material nucleic acid and protein levels of life. The life sciences have entered the era of “molecules†and produced a large number of new and interdisciplinary subjects. An important direction has had a major and profound impact on the development of epidemiology itself and on disease control.
Carpanenamase-producing Enterobacteriaceae (CPE) has emerged as a threat to hospitalized patients. Phenotypic test such as Modified hodge test was less sensitive and specific especially to detect blaNDM-1 which is the most predominant genotype in this region. Nucleic acid amplification technology offers improved specificity and sensitivity. Failed amplification due to the presence of inhibitors is a limitation. In this study, we tried to use previous method described by Villumseen et al with some modification using another DNA extraction kit. Methods: Ten mls of sterile whole blood taken from nearly expired blood bag from blood bank was spiked with 200 μl of 0.5mcFarland bacterial suspension from thirty-six confirmed isolates of blaNDM-1 carbapenamase-producing Klebsiella pneumoniae in an aerobic Bactec Plus and incubated until the growth was detected. The blood specimen was subjected to DNA extraction method using Macherey-Nachel, Nucleospin® Blood QuickPure followed with multiplex PCR. Results: Out of the 36 isolates, 12 isolates revealed blaNDM-1 , 9 isolates revealed blaNDM-1 and blaOXA-48, 7 isolates revealed blaNDM-1, blaVIM and blaKPC genotypes that were amplified at cycle threshold of less than 30. Another 8 isolates could not pick up any genotypes possibly due to pipetting error as all the internal control were amplified. Eight true negative gram negative isolates underwent same procedure and none amplified at a cycle threshold less than 30. Conclusion: This modified method was proved to give a high yield of CPE genotypes with the cycle threshold was set at less than or equal to 30 and able to overcome the presence of PCR inhibitors.