Application and evaluation of nucleic acid sequence-based amplification, PCR and cryptococcal antigen test for diagnosis of cryptococcosis

Background Cryptococcosis is a major opportunistic invasive mycosis in immunocompromised patients, but it is also increasingly seen in immunocompetent patients. In the early stages of cryptococcosis, limitations of the detection method may hinder the diagnosis. A molecular diagnostic technique based on nucleic acid sequence-based amplification (NASBA) method was developed to fulfil the need for efficient diagnosis of cryptococcosis. Methods We compared the diagnostic performance of NASBA, PCR and cryptococcal antigen (CrAg) test (colloidal gold method) in clinical samples from 25 cryptococcosis patients (including 8 cryptococcal meningoencephalitis and 17 pulmonary cryptococcosis) who were categorized as proven cases (n = 10) and probable cases (n = 15) according to the revised EORTC/MSG definitions. 10 patients with non-Cryptococcus infection and 30 healthy individuals were categorized as control group. Results The lowest detection limit of NASBA was 10 CFU/mL, and RNA of non-target bacteria or fungi was not amplified. The sensitivity of NASBA, PCR and colloidal gold method was 92.00% (95% CI 72.50–98.60%), 64.00% (95% CI 42.62–81.29%), 100.00% (95% CI 83.42–100.00%), and the specificity was 95.00% (95% CI 81.79–99.13%), 80.00% (95% CI 63.86–90.39%) and 82.50% (95% CI 66.64–92.11%) respectively. The highest specificity (97.50%), accuracy (95.38%) and k value (0.90) were achieved when both NASBA and colloidal gold results were positive. Conclusions NASBA is a new alternative detection method for cryptococcosis which is both accurate and rapid without expensive equipment and specialised personnel. It may be used as a tool for confirming current infection as well as monitoring the effectiveness of antifungal treatment. The use of NASBA to detect Cryptococcus RNA in blood samples is of great significance for the diagnosis of pulmonary cryptococcosis. The combination of NASBA and colloidal gold can improve the diagnostic accuracy of cryptococcosis.

RANCANGAN PRIMER UNTUK DETEKSI VIRUS DENGUE SEROTIPE DENV-3 DAN DENV-4 DENGAN METODE NASBA DAN LFIA

Simple, fast, and accurate early detection is expected to reduce the mortality rate due to dengue infection. The dengue virus RNA detection method using Nucleic Acid Sequence-Based Amplification (NASBA) is an alternative method that can reduce the use of a thermocycler. The detection of NASBA amplicons was carried out using the Lateral Flow Assay (LFIA). This study was conducted to prove the effectiveness of the new primer design to detect dengue virus serotypes DENV-3 and DENV-4. In addition, this study was also conducted to measure the sensitivity and specificity of the LFIA method to detect dengue virus serotypes DENV-3 and DENV-4. The initial stage of this research was the isolation of dengue virus RNA from C6/36 cell cultures, then proceeded to design primers for NASBA and LFIA probes. NASBA reactions were performed on dengue virus serotypes DENV-3 and DENV-4 and DENV-4. The NASBA reaction products were then visualized on LFIA and agarose gel electrophoresis. The NASBA method with a new primary design can be used for the detection of dengue virus serotypes DENV-3 and DENV-4 as evidenced by electrophoresis results bands at 196 bp and 144 bp. The LFIA method with probe design can be used for the detection of dengue virus serotype DENV-3 and DENV-4 dengue virus serotype with a positive line on the test line on LFIA. The LFIA method for the detection of the dengue virus has a sensitivity up to a concentration of 10-3 (1 g/μl). The results of this study indicate that the newly designed primers are specific and sensitive for DENV-3 and DENV-4 dengue virus serotypes detection. Abstrak Deteksi dini yang sederhana, cepat dan akurat diharapkan dapat mengurangi tingkat kematian akibat infeksi dengue. Metode deteksi RNA virus dengue dengan Nucleic Acid Sequence-Based Amplification (NASBA) merupakan metode alternatif yang dapat mengurangi penggunaan thermocycler. Deteksi amplikon hasil NASBA dilakukan dengan Lateral Flow Assay (LFIA). Studi ini dilakukan untuk membuktikan efektivitas desain baru primer untuk mendeteksi virus dengue serotipe DENV-3 dan DENV-4. Di samping itu, studi ini juga dilakukan untuk mengukur sensitivitas dan spesifisitas metode LFIA untuk mendeteksi virus dengue serotype DENV-3 dan DENV-4. Tahap awal penelitian ini adalah isolasi RNA virus dengue dari kultur sel C6/36, kemudian dilanjutkan dengan merancang primer untuk NASBA dan probe LFIA. Reaksi NASBA dilakukan pada virus dengue serotipe DENV-3 dan DENV-4 dan DENV-4. Produk reaksi NASBA kemudian divisualisasikan pada LFIA dan elektroforesis gel agarosa. Metode NASBA dengan desain primer baru dapat digunakan untuk deteksi virus dengue serotipe DENV-3 dan DENV-4 yang dibuktikan oleh pita hasil elektroforesis pada 196 bp dan 144 bp. Metode LFIA dengan desain probe dapat digunakan untuk deteksi virus dengue serotipe DENV-3 dan DENV-4 serotipe virus dengue dengan garis positif pada garis uji pada LFIA. Metode LFIA untuk deteksi virus dengue memiliki sensitivitas hingga konsentrasi 10-3 (1 μg/μl). Hasil penelitian ini menunjukkan bahwa primer baru yang dirancang bersifat spesifik dan sensitif untuk deteksi serotipe virus dengue DENV-3 dan DENV-4.

Gold–Oligonucleotide Nanoconstructs Engineered to Detect Conserved Enteroviral Nucleic Acid Sequences

Enteroviruses are ubiquitous mammalian pathogens that can produce mild to life-threatening disease. We developed a multimodal, rapid, accurate and economical point-of-care biosensor that can detect nucleic acid sequences conserved amongst 96% of all known enteroviruses. The biosensor harnesses the physicochemical properties of gold nanoparticles and oligonucleotides to provide colourimetric, spectroscopic and lateral flow-based identification of an exclusive enteroviral nucleic acid sequence (23 bases), which was identified through in silico screening. Oligonucleotides were designed to demonstrate specific complementarity towards the target enteroviral nucleic acid to produce aggregated gold–oligonucleotide nanoconstructs. The conserved target enteroviral nucleic acid sequence (≥1 × 10−7 M, ≥1.4 × 10−14 g/mL) initiates gold–oligonucleotide nanoconstruct disaggregation and a signal transduction mechanism, producing a colourimetric and spectroscopic blueshift (544 nm (purple) > 524 nm (red)). Furthermore, lateral-flow assays that utilise gold–oligonucleotide nanoconstructs were unaffected by contaminating human genomic DNA, demonstrated rapid detection of conserved target enteroviral nucleic acid sequence (<60 s), and could be interpreted with a bespoke software and hardware electronic interface. We anticipate that our methodology will translate in silico screening of nucleic acid databases to a tangible enteroviral desktop detector, which could be readily translated to related organisms. This will pave the way forward in the clinical evaluation of disease and complement existing strategies to overcome antimicrobial resistance.

The therapeutic application of a nucleic acid sequence to patients' diseased organs is currently available.

The therapeutic application of a nucleic acid sequence to patients' diseasedorgans or tissues is currently being studied in a Phase 1/2 human trial forfamilial hypercholesterolemic homozygosity. In the year 2025, the FDA has fiveto ten green drugs for clinical production, with proven progress. Luxturn andZolgensma have paved the way for a new method for treatment of hereditaryRPE65 and SMN-deficient retinal and spinal muscular dystrophy. It seems thatrAAV vectors are a hard organ to target, and therefore allow therapeutic genetherapy in the heart to be a concern. The discovery of other cardiac targets suchas S100A1/GRK2 inhibitors could be wise, although simple/efficient deliverysystems are being developed. The CRIS-dependent hypertrophiccardiomyopathy (mg) caused by the MYBPC3 mutation may be corrected usinghuman germline CRIS9 in stem cells, however. The investigation of non-codingDNA and epigenetics has increased the number of treatments available. Nucleictreatments can be used as a first-line treatment if pharmacotherapotherapieshave failed.

Ultrasensitive version of nucleic acid sequence-based amplification (NASBA) utilizing nicking and extension chain reaction system

Nucleic acid sequence-based amplification (NASBA) is a transcription-based isothermal amplification technique especially designed for the detection of RNA targets. The NASBA basically relies on the linear production of T7 RNA...