Development, evaluation of the PNA RT-LAMP assay for rapid molecular detection of SARS-CoV-2

Dual-labeled PNA probe used RT-LAMP molecular rapid assay targeting SARS-CoV-2 ORF1ab and N genes was developed, and the analytical, clinical performances for detection of SARS-CoV-2 RNA extracted from clinical nasopharyngeal swab specimens were evaluated in this study. Data showed that this assay is highly specific for SARS-CoV-2, and the absolute detection limit is 1 genomic copy per microliter of viral RNA which can be considered to be comparable to gold-standard molecular diagnostic method real-time reverse transcriptase PCR. Both clinical sensitivity and specificity against a commercial real-time RT-PCR assay were determined as identical. In conclusion, the PNA RT-LAMP assay showed high analytical and clinical accuracy which are identical to real-time RT-PCR which has been routinely used for the detection of SARS-CoV-2.

Development, Evaluation of the PNA RT-LAMP Assay for Rapid Molecular Detection of SARS-CoV-2

Dual-labeled PNA probe used RT-LAMP molecular rapid assay targeting SARS-CoV-2 ORF1ab and N genes was developed, and the analytical, clinical performances for detection of SARS-CoV-2 RNA extracted from clinical nasopharyngeal swab specimens were evaluated in this study. Data showed that this assay is highly specific for SARS-CoV-2, and the absolute detection limit is 1 genomic copy per microliter of viral RNA which can be considered to be comparable to gold-standard molecular diagnostic method real-time reverse transcriptase PCR. Both clinical sensitivity and specificity against a commercial real-time RT-PCR assay were determined as identical. In conclusion, the PNA RT-LAMP assay showed high analytical and clinical accuracy which are identical to real-time RT-PCR which has been routinely used for the detection of SARS-CoV-2.

Development and Validation of a High-Performance Thin-Layer Chromatographic Method for Determination of Ofloxacin Residues on Pharmaceutical Equipment Surfaces

An HPTLC method with densitometric quantification using fluorescence at 313 nm was developed and validated for the determination of ofloxacin residue in controlling pharmaceutical equipment cleanliness. Simulated samples at a residue level of 1 mg/m2 were prepared by spreading the calculated amount of ofloxacin solution on 1, 5, and 10 dm2 stainless steel surfaces. After evaporation of the solvent, the residue was removed by two ethanol wetted cotton swabs, which were thereafter extracted with the mixture of ethanol and Na2EDTA–water solution at pH 11 for 15 min with sonication. The extract and standards were applied on HPTLC silica gel 60 plates and then developed in a horizontal developing chamber from both sides using ethanol-conc. ammonia (4 + 1, v/v) as the mobile phase. The mean recovery (n = 6) at 1 mg/m2 from 1, 5, and 10 dm2 was 95.3, 88.6, and 89.7% with the CV values 3.78, 4.41, and 4.97%, respectively. The absolute detection limit was 0.6 ng and the quantitation limit was 2 ng, but it was shown that these can be improved by immersion of the developed plate into a solution of liquid paraffin–n-hexane (1 + 2, v/v) to approximately 0.25 and 0.9 ng, respectively. The LOD of the method using detection without paraffin–n-hexane was 3, 0.6, and 0.3 μg/m2 by swabbing 1, 5, and 10 dm2, respectively. The method can be applied to routine control of pharmaceutical equipment cleanliness by sampling from stainless steel surface areas of 1 to 10 dm2 with acceptable residue limit/surface of 1 mg/m2.

SYBR Green Real-Time PCR Used to Detect Celery in Food

Celery was found to provoke human allergenic response in some countries. Labeling of celery ingredients was required by the European Union, and the threshold set at 10 mg/kg (0.001%). In our study, a celery mannitol transporter (Mat3) gene-based detection method was established by means of SYBR Green real-time PCR technique. No cross-reactivity was found between celery and the other food materials. Absolute detection limit (LODa), relative detection limit (LODr), and practical detection limit (LODp) of the method were determined through experiments on pure celery DNA, DNA mix, and spiked food samples. The method was able to detect 0.001 raw food sample and 0.01 heated food sample. The utility of the method was confirmed by the investigation of 13 commercial foods.

Development and Validation of a Thin-Layer Chromatographic Method for Determination of Chloramphenicol Residues on Pharmaceutical Equipment Surfaces

A thin-layer chromatographic (TLC) method with densitometric quantitation using the absorption reflectance mode at 280 nm was developed and validated for the determination of chloramphenicol residues in controlling pharmaceutical equipment cleanliness. Simulated samples at residue levels 0.5, 1, and 1.2 mg/m2 were prepared by spreading the calculated amount of chloramphenicol solution on a 10 dm2 stainless steel surface. After evaporation of the solvent, the residue was removed by 2 methanol-wetted cotton swabs, which were then extracted with methanol. The extract was applied on a high-performance TLC (HPTLC) silica gel F254 plate together with standards ranging from 10 to 60 ng. Plates were developed in a horizontal developing chamber from both sides (36 applications per plate) by using n-hexane–ethyl acetate (35 + 65, v/v) as developing solvent. The mean recovery (n = 6) at 1 mg/m2 was 95.8%, and the coefficient of variation was 5.8%. The absolute detection limit was 3 ng, and the quantitation limit 10 ng. The method detection limit was 0.3 mg/m2 by swabbing 2.5 dm2 and 0.075 mg/m2 by swabbing 10 dm2. Chloramphenicol was stable on the plate 2 h before and 24 h after development. Additionally, it was stable during 7 days storage on the cotton swabs in the solvent at room temperature and in diluted standard solution stored in darkness at 4°C. The method can be applied to routine control of pharmaceutical equipment cleanliness by sampling from the stainless steel surface areas of 2.5 to 10 dm2, and an acceptable residue limit of 1 mg/m2.

Affinity Purification of DNA and RNA from Environmental Samples with Peptide Nucleic Acid Clamps

ABSTRACT Bispeptide nucleic acids (bis-PNAs; PNA clamps), PNA oligomers, and DNA oligonucleotides were evaluated as affinity purification reagents for subfemtomolar 16S ribosomal DNA (rDNA) and rRNA targets in soil, sediment, and industrial air filter nucleic acid extracts. Under low-salt hybridization conditions (10 mM NaPO4, 5 mM disodium EDTA, and 0.025% sodium dodecyl sulfate [SDS]) a PNA clamp recovered significantly more target DNA than either PNA or DNA oligomers. The efficacy of PNA clamps and oligomers was generally enhanced in the presence of excess nontarget DNA and in a low-salt extraction-hybridization buffer. Under high-salt conditions (200 mM NaPO4, 100 mM disodium EDTA, and 0.5% SDS), however, capture efficiencies with the DNA oligomer were significantly greater than with the PNA clamp and PNA oligomer. Recovery and detection efficiencies for target DNA concentrations of ≥100 pg were generally >20% but depended upon the specific probe, solution background, and salt condition. The DNA probe had a lower absolute detection limit of 100 fg of target (830 zM [1 zM = 10−21 M]) in high-salt buffer. In the absence of exogenous DNA (e.g., soil background), neither the bis-PNA nor the PNA oligomer achieved the same absolute detection limit even under a more favorable low-salt hybridization condition. In the presence of a soil background, however, both PNA probes provided more sensitive absolute purification and detection (830 zM) than the DNA oligomer. In varied environmental samples, the rank order for capture probe performance in high-salt buffer was DNA > PNA > clamp. Recovery of 16S rRNA from environmental samples mirrored quantitative results for DNA target recovery, with the DNA oligomer generating more positive results than either the bis-PNA or PNA oligomer, but PNA probes provided a greater incidence of detection from environmental samples that also contained a higher concentration of nontarget DNA and RNA. Significant interactions between probe type and environmental sample indicate that the most efficacious capture system depends upon the particular sample type (and background nucleic acid concentration), target (DNA or RNA), and detection objective.

Ion localization in Cells by Electron Probe X-Ray Microanalysis (Epxma) Depends on Cryopreparation

Elements in biological cells can be localized by EPXMA. The most attractive approach of this method with respect to detection limit and spatial resolution is scanning transmission electron microscopy combined with energy dispersive x ray microanalysis of ultrathin (approximately 100 nm thick) freeze-dried cryosections. The detection limit, here determined by scanning an electron beam of 1.3 nA for 2 min over freeze-dried cryosections from frozen standard solutions was found to be about 10 mMol/kg dry weight for all elements with the atomic number Z higher than 12. Due to the Be window in the x ray detector the detection limit was 20 mMol/kg dry weight for Mg (Z = 12) and 30 mMol/kg dry weight for Na (Z = 11). The lateral analytical resolution was less than 50 nm, limited by section thickness. In most cells 10 mMol/kg dry weight correspond to an absolute detection limit of .500 atoms within a volume of 100×50×50 nm3.

Determination of Sodium Dioctylsulfosuccinate in Dry Beverage Bases by Liquid Chromatography with Post-Column Ion-Pair Extraction and Absorbance Detection

Sodium dioctylsulfosuccinate (DSS) is extracted as an ion pair with methylene blue from finished drinks prepared from dry beverage bases. The complex is quantitatively determined colorimetrically in chloroform-acetone solution by a standard procedure. DSS is specifically identified by analyzing an aliquot of the extract by reverse phase liquid chromatography (LC). The compound is detected by using a simple post-column dynamic extraction system in which DSS is extracted from the aqueous mobile phase into chloroform as a methylene blue ion pair. The chloroform phase passes through the absorbance detector for measurement at 546 nm (filter detector). The absolute detection limit was 5-10 ng DSS, while in beverage bases as low as 0.1 μg/g was detected. Extraction of the beverage bases with mobile phase followed by filtration and direct LC analysis with the described system was also successful, although not evaluated on a routine basis.

Determination of Cyclopiazonic Acid in Peanuts and Corn by Thin Layer Chromatography

A thin layer chromatographic system including densitometry has been developed for determining cyclopiazonic acid in peanuts and corn. Samples are extracted with methanol-chloroform (20 + 80); the extract is stripped of most interferences by partitioning with aqueous sodium bicarbonate followed by acidification and repartitioning with chloroform. After thin layer chromatography and derivatization with dimethylaminobenzaldehyde- HCl spray, the toxin is quantitated by reflection densitometry at 540 nm. The recovery of cyclopiazonic acid averages 90% for peanuts and 85% for corn. The absolute detection limit is 25 ng per spot which translates to a detection limit of 125 μg/kg for a 50 g sample.

Anion-exchange chromatographic determination of bromide in serum.

In this liquid-chromatographic method for the determination of bromide in human serum, an important and unique feature is pre-analysis ultrafiltration of the sample, which effectively removes proteins. The chromatographic system consists of an anion-exchange column (Whatman Partisil SAX 10-micron particles), an isocratic phosphate-buffered mobile phase, and ultraviolet detection at 195 nm. The absolute detection limit, 0.6 ng of bromide, makes this method the most sensitive assay for serum bromide yet reported. The day-to-day CV ranged from 1.6% to 4.9%. Analytical recovery of bromide added to serum samples ranged from 96.2% to 108.8%. Data on bromide in serum are presented for adults and newborns and for adults given bromide orally.