Nucleic Acid Amplification Techniques Research Articles

Environmental surveillance of Cryptosporidium and Giardia in surface supply water and treated sewage intended for reuse from an urban area in Brazil

Environmental monitoring of protozoa, with the potential to trigger diseases, is essential for decision-making by managing authorities and for the control of water surveillance. This study aimed to detect and quantify Cryptosporidium oocysts and Giardia cysts in surface water for drinking water supply and treated sewage for reuse in the city of São Paulo. Samples collected bimonthly for one year were concentrated using the USEPA 1623.1 and 1693 methods for surface water and treated effluents, respectively. Immunofluorescence and nucleic acid amplification techniques were used to detect and quantify (oo)cysts. The cloning technique followed by sequencing and phylogenetic analyses were performed to characterize species and genotypes. The immunofluorescence detected Cryptosporidium spp. and Giardia spp. in 69.2% (9/13) and 100% (13/13) of the surface water samples (0.1–41 oocysts/L and 7.2–354 cysts/L, respectively). In the reuse samples, 85.7% (12/14) were positive for both protozoa and the concentrations varied from 0.4 to 100.6 oocysts/L and 1.2 and 93.5 cysts/L. qPCR assays showed that 100% of surface water (0.1–14.6 oocysts/L and 0.3–639.8 cysts/L) and reused samples (0.1–26.6 oocysts/L and 0.3–92.5 cysts/L) were positive for both protozoa. Species C. parvum, C. hominis, and C. muris were identified using the 18S rRNA gene, demonstrating anthroponotic and zoonotic species in the samples. Multilocus SSU rRNAanalyses of the SSU rRNA, tpi, and gdh genes from Giardia intestinalis identified the AII, BII, and BIV assemblages, revealing that contamination in the different matrices comes from human isolates. The study showed the circulation of these protozoa in the São Paulo city area and the impairment of surface water supply in metropolitan regions impacted by the discharge of untreated or inadequately treated sewage regarding the removal of protozoa, emphasizing the need to implement policies for water safety, to prevent the spread of these protozoa in the population.

Diagnostic accuracy of point-of-care tests for acute respiratory infection: a systematic review of reviews.

Acute respiratory infections are a common reason for consultation with primary and emergency healthcare services. Identifying individuals with a bacterial infection is crucial to ensure appropriate treatment. However, it is also important to avoid overprescription of antibiotics, to prevent unnecessary side effects and antimicrobial resistance. We conducted a systematic review to summarise evidence on the diagnostic accuracy of symptoms, signs and point-of-care tests to diagnose bacterial respiratory tract infection in adults, and to diagnose two common respiratory viruses, influenza and respiratory syncytial virus. The primary approach was an overview of existing systematic reviews. We conducted literature searches (22 May 2023) to identify systematic reviews of the diagnostic accuracy of point-of-care tests. Where multiple reviews were identified, we selected the most recent and comprehensive review, with the greatest overlap in scope with our review question. Methodological quality was assessed using the Risk of Bias in Systematic Reviews tool. Summary estimates of diagnostic accuracy (sensitivity, specificity or area under the curve) were extracted. Where no systematic review was identified, we searched for primary studies. We extracted sufficient data to construct a 2 × 2 table of diagnostic accuracy, to calculate sensitivity and specificity. Methodological quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies version 2 tool. Where possible, meta-analyses were conducted. We used GRADE to assess the certainty of the evidence from existing reviews and new analyses. We identified 23 reviews which addressed our review question; 6 were selected as the most comprehensive and similar in scope to our review protocol. These systematic reviews considered the following tests for bacterial respiratory infection: individual symptoms and signs; combinations of symptoms and signs (in clinical prediction models); clinical prediction models incorporating C-reactive protein; and biological markers related to infection (including C-reactive protein, procalcitonin and others). We also identified systematic reviews that reported the accuracy of specific tests for influenza and respiratory syncytial virus. No reviews were found that assessed the diagnostic accuracy of white cell count for bacterial respiratory infection, or multiplex tests for influenza and respiratory syncytial virus. We therefore conducted searches for primary studies, and carried out meta-analyses for these index tests. Overall, we found that symptoms and signs have poor diagnostic accuracy for bacterial respiratory infection (sensitivity ranging from 9.6% to 89.1%; specificity ranging from 13.4% to 95%). Accuracy of biomarkers was slightly better, particularly when combinations of biomarkers were used (sensitivity 80-90%, specificity 82-93%). The sensitivity and specificity for influenza or respiratory syncytial virus varied considerably across the different types of tests. Tests involving nucleic acid amplification techniques (either single pathogen or multiplex tests) had the highest diagnostic accuracy for influenza (sensitivity 91-99.8%, specificity 96.8-99.4%). Most of the evidence was considered low or very low certainty when assessed with GRADE, due to imprecision in effect estimates, the potential for bias and the inclusion of participants outside the scope of this review (children, or people in hospital). Currently evidence is insufficient to support routine use of point-of-care tests in primary and emergency care. Further work must establish whether the introduction of point-of-care tests adds value, or simply increases healthcare costs. This article presents independent research funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme as award number NIHR159948.

Aptamer-based Strategies for COVID-19 Detection and Treatment: A Systematic Review Study

Background: Aptamer-based strategies have emerged as promising tools for the detection and treatment of COVID-19, offering advantages such as high specificity, sensitivity, and versatility. This systematic review aims to evaluate the effectiveness and innovation of aptamer-based approaches for COVID-19 detection and treatment. Methods: Following the guidelines of the Cochrane Handbook for Systematic Reviews and the PRISMA 2020 guidelines, a systematic search was conducted across multiple databases up to 2024. The search included studies that utilized aptamers for the diagnosis or therapy of COVID-19. Screening and selection of studies were performed independently by two reviewers, with any disagreements resolved by a third reviewer. Data were extracted regarding study characteristics, aptamer details, and outcomes. Results: In our systematic review, 98 studies from an initial pool of 1541 records met the inclusion criteria for analysis. Aptamers, single-stranded DNA or RNA molecules with unique threedimensional (3D) structures, were extensively explored for COVID-19 detection and treatment. Various aptamer-based assays, including electrochemical sensors, surface plasmon resonance (SPR) biosensors, and lateral flow assays, demonstrated high sensitivity and specificity in detecting SARSCoV- 2 in clinical samples such as saliva, nasal swabs, and wastewater. Several aptamer structures targeting viral proteins like the spike and nucleocapsid proteins were employed. Nucleic Acid Amplification Techniques (NAATs) utilizing aptamers, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based and Loop-mediated Isothermal Amplification (LAMP) assays, showed exceptional sensitivity in detecting viral genetic material. Aptamer-based therapeutic approaches showed potential by blocking viral protein activity or serving as delivery vehicles for therapeutic agents like small interfering RNAs (siRNAs). Despite their advantages, aptamer technologies face limitations such as susceptibility to nuclease degradation and rapid renal clearance, highlighting the need for further optimization. Conclusion: Aptamer-based strategies present promising avenues for COVID-19 detection and treatment. These approaches offer advantages such as high sensitivity, specificity, and rapid detection, making them valuable tools in combating the COVID-19 pandemic. Further research and development are warranted to optimize aptamer-based strategies for widespread application in clinical settings.

New Approaches to Plant Pathogen Detection and Disease Diagnosis.

Detecting plant pathogens and diagnosing diseases are critical components of successful pest management. These key areas have undergone significant advancements driven by breakthroughs in molecular biology and remote sensing technologies within the realm of precision agriculture. Notably, nucleic acid amplification techniques, with recent emphasis on sequencing procedures, particularly next-generation sequencing, have enabled improved DNA or RNA amplification detection protocols that now enable previously unthinkable strategies aimed at dissecting plant microbiota, including the disease-causing components. Simultaneously, the domain of remote sensing has seen the emergence of cutting-edge imaging sensor technologies and the integration of powerful computational tools, such as machine learning. These innovations enable spectral analysis of foliar symptoms and specific pathogen-induced alterations, making imaging spectroscopy and thermal imaging fundamental tools for large-scale disease surveillance and monitoring. These technologies contribute significantly to understanding the temporal and spatial dynamics of plant diseases.

Genomic analysis of BAX and Bcl-2 gene mutations in human papilloma virus-associated squamous cell carcinoma of the cervix

PurposeThe aim of the current study was to molecularly determine Bcl-2 and BAX gene mutations in HPV-associated squamous cell carcinoma of the cervix.MethodsFormalin-fixed, paraffin-embedded tissue blocks, all consisting of squamous cell carcinoma of the cervix, were used for this study. The nucleic acid amplification technique and various steps for DNA sequencing, including DNA extraction and polymerase chain reaction, were used.ResultsMutations were detected in the Bcl-2 gene of patients with squamous cell carcinoma of the cervix in the 10–860 bp region, while BAX gene mutations were detected in the 10–320 bp region. The nucleotide mutations in the Bcl-2 gene were A > G (50%), C > T (33.33%), and G > A > T (16.67%), while the BAX gene mutations were A > (16.67%), T > (16.67%), G > (16.67%), A > C (16.67%), T > G (16.67%), and T > C (16.67%). The mutations in the BAX gene were Indel (50%), Transversion (33.4%), and Transition (16.6%), while only the Transition mutation (100%) was detected in the Bcl-2 gene. The functional mutations in the BAX gene were only missense mutations (100%), but in the Bcl-2 gene, the functional mutations were missense (50%) and silent (50%) mutations.ConclusionOur findings revealed genomic mutations of different types and frequencies in the BAX and Bcl-2 genes in squamous cell carcinoma of the cervix, which should encourage further research to better understand these mutations and exploit them for clinical use.

Prevalence of Human Papillomavirus Infection and its Association with the Risk of Cervical Cancer among Hiv-Positive Women in Plateau State, North-Central Nigeria

Study’s Novelty/Excerpt This study investigates the prevalence and risk factors of HPV among HIV-infected women in Plateau State, Nigeria, highlighting a significant correlation between low CD4+ counts, high viral loads, and increased HPV infection rates. By utilizing comprehensive diagnostic methods including ELISA for HPV detection and cytology for cervical abnormalities, the research offers robust data linking immunosuppression and HPV-related cervical pathology in a high-risk population. The findings emphasize the urgent need for targeted interventions to improve sexual health behaviors and further research on how low immunity accelerates cervical cancer progression in both HIV-positive women and the broader population. Full Abstract Human papillomavirus (HPV) is one of the most common sexually transmitted infections (STI) associated with cervical, uterine, and anogenital cancers. Persistent infection with HPV is associated with abnormal cervical cells, which can develop into cervical cancer if left untreated. Human papillomaviruses are the first viruses to have been acknowledged to prompt carcinogenesis, and they are linked with cancers of the uterine cervix, anogenital tumours, and head and neck malignancies. A hospital-based study of HIV-infected women across the three senatorial zones of Plateau State, Nigeria, was conducted between November 2018 to November 2020. Ethical approval for the study was first obtained from the ethical committee of Plateau State Specialist Hospital Jos, and informed consent to participate in the research was also obtained from each participant. HIV status confirmation was first done through standard rapid test procedures, followed by cytology testing via the Pap smear procedure to detect any precancerous or malignant changes in the cervix. Subsequent detection of HPV utilized the ELISA procedure, while CD4+ cell count and viral load estimations were done using flow cytometry and nucleic acid amplification techniques, respectively. Questionnaires were administered to obtain information on cervical cancer risk factors and clinical presentations. The overall prevalence of HPV was 28% among HIV-infected women. More HPV infection (31.9%) occurred in women with low CD4+ count (0-200 cells/mm3), and also highest (50.0%) among women with the highest HIV viral load (>100 copies/mL). The possible risk factors identified in this study include multiple sexual partnering, low condom usage, and coinfection with other STIs, among others. In conclusion, this study identified a high HPV prevalence, low CD4+ counts, and coinfection with other STIs among high-risk populations (HIV-infected women). We, therefore, recommend improved sexual behaviours and further research on the impact of low immunity on the rate of progression of cervical abnormality to cervical cancer, not just in HIV-positive women but in the general population.

Dual-mode SERS-ECL biosensor for robust detection of circulating miRNAs based on in-situ synthesized probes

A novel dual-mode biosensor integrating surface-enhanced Raman spectroscopy (SERS) and electrochemiluminescence (ECL) was developed for the reliable detection of acute myocardial infarction (AMI) related circulating miRNAs. The DNA walker nucleic acid amplification technique was employed for signal amplification and facilitated in-situ synthesis of ECL luminescent clusters and SERS probes. The alkynyl-modified silver nanocluster (AgNC) template strand and the azido-modified DNA probe on the electrode were connected through linker release facilitated by the continuous rolling and cutting action of the DNA walker on the spherical substrate. Under Cu+ catalysis, the alkynyl and azide groups at the junction undergo conversion into triazole molecules, which can serve as SERS probes. Subsequently, AgNCs were synthesized using a template method, with SERS signals emitted due to plasma resonance effects of triazole molecules situated between Au nanoparticles and AgNCs. The developed dual-mode analysis platform efficiently minimizes the potential for signal leakage, significantly corrects systematic errors, improves detection accuracy and sensitivity, and facilitates dependable identification of circulating miRNA. The detection of miRNA-133a exhibited a linear relationship within the concentration range of 100 aM to 1 nM, with detection limits of 32 aM and 0.17 fM in ECL and SERS modes, respectively. The SERS-ECL dual-mode detection platform may provide forward-looking ideas for the field of biosensing and clinical analysis.

Detection of the thaumatin-like protein gene from Brassica rapa var. oleifera in honey with the Proofman-LMTIA method

ABSTRACT Honey adulteration has been on the rise with the increasing demand for high-quality honey. The ladders-shape melting temperature isothermal amplification (LMTIA) was a newly developed nucleic acid amplification technique, and the aim of this study was to establish a method combining the LMTIA with the proofreading enzyme-mediated probe cleavage (Proof-man probe) for detecting the thaumatin-like protein (TLP) genes of Brassica rapa var. oleifera in honey. The LMTIA primers was selected as a target to be designed, the temperature of the Proof-man LMTIA reaction was optimized, and the specificity, sensitivity, and applicability to the honey samples were determined. The results showed that the Proof-man LMTIA method was highly specific with a detection sensitivity of 30-40 copies genomic DNA/reaction at the optimal temperature of 60°C, and the amplification could be completed within 20 min. This method can detect the adulteration of low-price B. rapa honey into high-price honey.

Evaluating Commercial Loop-Mediated Isothermal Amplification Master Mixes for Enhanced Detection of Foodborne Pathogens.

Loop-mediated isothermal amplification, LAMP, is nowadays the most popular isothermal nucleic acid amplification technique, and as such, several commercial, ready-to-use master mixes have flourished. Unfortunately, independent studies to determine their performance are limited. The current study performed an independent evaluation of the existing ready-to-use commercial LAMP master mixes WarmStart® LAMP Kit, LavaLAMP™ DNA Master Mix, Saphir Bst Turbo GreenMaster, OptiGene Fast Master Mix ISO-004, and SynLAMP Mix. To reduce bias, three different genes, namely ttr (Salmonella spp.), rfbE (E. coli O157), and hly (Listeria monocytogenes), were targeted. The comparison was based on amplification speed, performance with decreasing DNA concentrations, and the effect of five typical LAMP reaction additives (betaine, DMSO, pullulan, TMAC, and GuHCl). Significant differences were observed among the different master mixes. OptiGene provided the fastest amplification and showed less detrimental effects associated with the supplements evaluated. Out of the chemicals tested, pullulan provided the best results in terms of amplification speed. It is noteworthy that the different additives impacted the master mixes differently. Overall, the current study provides insights into the performance of commercial LAMP master mixes, which can be of value for the scientific community to better select appropriate reagents when developing new methods.

Optical lateral flow assays in early diagnosis of SARS-CoV-2 infection.

So far, the 2019 novel coronavirus (COVID-19) is spreading widely worldwide. The early diagnosis of infection by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is essential to provide timely treatment and prevent its further spread. Lateral flow assays (LFAs) have the advantages of rapid detection, simple operation, low cost, ease of mass production, and no need for special devices and professional operators, which make them suitable for self-testing at home. This review focuses on the early diagnosis of SARS-CoV-2 infection based on optical LFAs including colorimetric, fluorescent (FL), chemiluminescent (CL), and surface-enhanced Raman scattering (SERS) LFAs for the detection of SARS-CoV-2 antigens and nucleic acids. The types of recognition components, detection modes used for antigen detection, labels employed in different optical LFAs, and strategies to improve the detection sensitivity of LFAs were reviewed. Meanwhile, LFAs coupled with different nucleic acid amplification techniques and CRISPR-Cas systems for the detection of SARS-CoV-2 nucleic acids were summarized. We hope this review provides research mentalities for developing highly sensitive LFAs that can be used in home self-testing for the early diagnosis of SARS-CoV-2 infection.

VLP-based model for the study of airborne viral pathogens.

The study and detection of airborne pathogens are hampered by the lack of appropriate model systems. In this work, we demonstrate that noninfectious virus-like particles (VLPs) represent attractive models to study airborne viral pathogens. Specifically, VLPs are readily prepared, are similar in size and composition to infectious viruses, and are amenable to highly sensitive nucleic acid amplification techniques.

Endonuclease Q as a robust enhancer for nucleic acid amplification

Isothermal nucleic acid amplification techniques are attracting increasing attention in molecular diagnosis and biotechnology. However, most existing techniques are complicated by the need for intricate primer design and numerous enzymes and primers. Here, we have developed a simple method, termed NAQ, that employs adding both endonuclease Q (EndoQ) and dUTP/dITP to conventional rolling circle amplification reactions to increase DNA amplification. NAQ does not require intricate primer design or DNA sequence-specific enzymes, and existing isothermal amplification techniques could be readily adapted to include both EndoQ and dUTP/dITP.

Utility of CBNAAT (GeneXpert MTB/RIF assay) in rapid diagnosis of extrapulmonary tuberculosis in a hepatobiliary tertiary center

Objectives Newer diagnostic techniques like cartridge-based nucleic acid amplification techniques (CB NAAT) need to be evaluated for extrapulmonary tuberculosis (EPTB), as being a paucibacillary condition, it is often underdiagnosed with conventional methods. We conducted this study to assess the utility of CB NAAT (GeneXpert MTB/RIF assay) in rapid diagnosis of extrapulmonary tuberculosis. Material and Methods Liver disease patients admitted from June 2019 to June 2020 were investigated for EPTB based on clinical and radiological suspicion. EPTB was diagnosed based on one of the following: (i) histological evidence of caseating granulomas; (ii) smear positivity for acid-fast bacilli; (iii) CB NAAT (GeneXpert MTB/RIF assay, Cepheid, USA). Results A total of 290 EPTB specimens received in the laboratory were included. The extrapulmonary samples that were received included body fluids (n = 143) which included pleural fluid, ascitic fluid, drain fluids, and pus aspirates, followed by biopsies (n = 82), lymph nodes (n = 43), urine (n = 19), and CSF (n = 3). GeneXpert MTB/RIF assay was positive in 10.3% (n = 30) samples, whereas negative in 89.7% (n = 260) samples. The overall sensitivity of GeneXpert MTB/RIF assay was 61.36% (95% CI 46.62%–74.28%), specificity 89.29% (95% CI 72.8%–96.29%), positive predictive value (PPV) 90% (95% CI 74.38%–96.54%), and negative predictive value (NPV) 59.52% (95% CI 44.49%–72.96%). Conclusion The GeneXpert MTB/RIF assay is a valuable tool for extrapulmonary tuberculosis. In addition to other tests like smear, culture GeneXpert MTB/RIF assay helps in the confirmation of diagnosis. Rapid diagnosis of tuberculosis with overall good sensitivity and specificity makes it a beneficial test.

Non-Microbiological Mycobacterial Detection Techniques for Quality Control of Biological Products: A Comprehensive Review.

Mycobacteria can be one of the main contaminants of biological products, and their presence can have serious consequences on patients' health. For this reason, the European Pharmacopoeia mandates the specific testing of biological products for mycobacteria, a critical regulatory requirement aimed at ensuring the safety of these products before they are released to the market. The current pharmacopeial reference, i.e., microbial culture method, cannot ensure an exhaustive detection of mycobacteria due to their growth characteristics. Additionally, the method is time consuming and requires a continuous supply of culture media, posing logistical challenges. Thus, to overcome these issues, pharmaceutical industries need to consider alternative non-microbiological techniques to detect these fastidious, slow-growing contaminating agents. This review provides an overview of alternative methods, which could be applied within a quality control environment for biological products and underlines their advantages and limitations. Nucleic acid amplification techniques or direct measurement of mycobacteria stand out as the most suitable alternatives for mycobacterial testing in biological products.

Towards real-time airborne pathogen sensing: Electrostatic capture and on-chip LAMP based detection of airborne viral pathogens

Considerable loss of life, economic slowdown, and public health risk associated with the transmission of airborne respiratory pathogens was underscored by the recent COVID-19 pandemic. Airborne transmission of zoonotic diseases such as the highly pathogenic avian influenza (HPAI) and porcine reproductive and respiratory syndrome virus (PRRSV) has caused major disruptions to domestic and global food security. Current ambient air pathogen monitoring systems involves the collection of air samples from indoor settings suspected of viral contamination, followed by subsequent processing of capture samples to determine the presence and species of airborne viral matter. Nucleic acid amplification techniques are considered the gold standard for pathogen diagnostics. Currently, the necessary extraction and purification of viral RNA from air collector systems prior to sample analysis is both time consuming and performed manually. A monitoring system with separate air sampling and biochemical detection procedures is prone to delay the response to emergent viral threats. In this paper, we present a pathogen monitoring system that overcomes these limitations related to extraction and purification of viral samples and lays the groundwork for a real-time monitor for airborne viral pathogens. We demonstrate a high flow electrostatic precipitator system, that uses small collection wells as counter electrodes for pathogen collection. Integrated reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) is used for detection of captured viral matter within wells. On-chip heating of collection wells is enabled by integrated planar heaters and small volumes of reagent (30 μL) directly to the collection wells. We present the design of such a system and show experimental results that demonstrate the use of this device for detection of aerosolized SARS-CoV-2 virus like particles (VLPs), a model pathogen for SARV-CoV-2.

Recombinase-Aided Loop-Mediated Isothermal Amplification on Human Plasmodium knowlesi.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that can amplify specific nucleic acids at a constant temperature (63-65°C) within a short period (<1 hour). In this study, we report the utilization of recombinase-aided LAMP to specifically amplify the 18S sRNA of Plasmodium knowlesi. The method was built on a conventional LAMP assay by inclusion of an extra enzyme, namely recombinase, into the master mixture. With the addition of recombinase into the LAMP assay, the assay speed was executed within a time frame of less than 28 minutes at 65°C. We screened 55 P. knowlesi samples and 47 non-P. knowlesi samples. No cross-reactivity was observed for non-P. knowlesi samples, and the detection limit for recombinase-aided LAMP was one copy for P. knowlesi after LAMP amplification. It has been reported elsewhere that LAMP can be detected through fluorescent readout systems. Although such systems result in considerable limits of detection, the need for sophisticated equipment limits their use. Hence, we used here a colorimetric detection platform for the evaluation of the LAMP assay's performance. This malachite green-based recombinase-aided LAMP assay enabled visualization of results with the naked eye. Negative samples were observed by a change in color from green to colorless, whereas positive samples remained green. Our results demonstrate that the LAMP assay developed here is a convenient, sensitive, and useful diagnostic tool for the rapid detection of knowlesi malaria parasites. This method is suitable for implementation in remote healthcare settings, where centralized laboratory facilities, funds, and clinicians are in short supply.

From Wet Mount to Nucleic Acid Amplification Techniques: Current Diagnostic Methods and Future Perspectives Based on Patenting of New Assays, Stains, and Diagnostic Images for Trichomonas vaginalis Detection

Trichomoniasis is the most common non-viral sexually transmitted infection (STI) in the world. The estimated global prevalence in 2016 was 156 million adults aged 15–49. However, these data are underestimated, since the most used diagnostic method is the wet mount, which has low sensitivity, the information regarding the estimated duration of infection is limited and there is evidence of undiagnosed asymptomatic cases in both sexes. Currently 80% of cases—including both sexes—are asymptomatic, which makes the disease silent and chronic in course, leading to complications. The aim of this review was to discuss the diagnostic methods for T. vaginalis detection that are currently available and applicable in the clinical laboratory routine. Overall, nucleic acid amplification techniques are the best option for T. vaginalis detection, with higher sensitivity and specificity than other tests. Although these techniques present higher cost, their implementation should be supported to ensure correct trichomoniasis diagnosis and treatment beyond contributing to questions on epidemiology and control.

Confirmatory Assay for Laboratory Diagnosis of Malaria Using Molecular Approach.

Prompt malarial treatment and surveillance is crucial for accurate diagnosis of Plasmodium Sp. Gold standard microscopic examination has been widely applied for diagnosis of malaria in most part of the endemic areas. But in case of submicroscopic and asymptomatic microscopic diagnosis is questioned. The study aims to develop a simple, cost effective & robust nucleic acid amplification technique for the detection of malaria parasite. Study population included 50 clinically diagnosed positive malaria patient samples from various pathological laboratories. Microscopy by preparing thick film was carried out of every sample for primary screening in the available facility of Surat Raktadan Kendra & Research Centre- Blood Bank. The conventional PCR (Polymerase Chain Reaction) was applied for genus-specific amplification targeting the 18S rRNA gene of Plasmodium. Agarose gel electrophoresis was used to separate and analyze the amplified PCR product using 2% Agarose gel. The study shows that nested PCR not only detected all microscopic positive samples, but also detected submicroscopic infections that were missed or misread by microscopy. Hence, the sensitivity of molecular based detection technique is proved to be more compared to microscopic examination.

Recombinase Polymerase Amplification assay for detection of the British root-knot nematode, Meloidogyne artiellia.

Recombinase polymerase amplification (RPA) is an isothermal in vitro nucleic acid amplification technique that has been adopted for simple, robust, rapid, reliable diagnostics of nematodes. In this study, the real-time RPA assay and RPA assay combined with lateral flow dipsticks (LF-RPA) have been developed targeting the ITS rRNA gene of the British root-knot nematode, Meloidogyne artiellia. The assay provided specific and rapid detection of this root-knot nematode species from crude nematode extracts without a DNA extraction step with a sensitivity of 0.125 second-stage juvenile (J2) specimen per a reaction tube for real-time RPA during 11 min and a sensitivity of 0.5 J2 specimens per a reaction tube for LF-RPA during 25 min. The RPA assays were validated with a wide range of non-target root-knot nematodes. The LF-RPA assay has great potential for nematode diagnostics in the laboratory having minimal available equipment.

Advances and Challenges in SARS-CoV-2 Detection: A Review of Molecular and Serological Technologies.

The urgent need for accurate COVID-19 diagnostics has led to the development of various SARS-CoV-2 detection technologies. Real-time reverse transcriptase polymerase chain reaction (RT-qPCR) remains a reliable viral gene detection technique, while other molecular methods, including nucleic acid amplification techniques (NAATs) and isothermal amplification techniques, provide diverse and effective approaches. Serological assays, detecting antibodies in response to viral infection, are crucial for disease surveillance. Saliva-based immunoassays show promise for surveillance purposes. The efficiency of SARS-CoV-2 antibody detection varies, with IgM indicating recent exposure and IgG offering prolonged detectability. Various rapid tests, including lateral-flow immunoassays, present opportunities for quick diagnosis, but their clinical significance requires validation through further studies. Challenges include variations in specificity and sensitivity among testing platforms and evolving assay sensitivities over time. SARS-CoV-2 antigens, particularly the N and S proteins, play a crucial role in diagnostic methods. Innovative approaches, such as nanozyme-based assays and specific nucleotide aptamers, offer enhanced sensitivity and flexibility. In conclusion, ongoing advancements in SARS-CoV-2 detection methods contribute to the global effort in combating the COVID-19 pandemic.