Reverse Transcription Loop-Mediated Isothermal Amplification Assay with High Sensitivity to Rapid Detection of Viable Salmonella in Foods

Background: Salmonella is one of the main foodborne bacterial pathogens, causing diseases and death. The study used reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect Salmonella. Objectives: To design six primers and detect Salmonella using RT-LAMP to facilitate the rapid detection of pathogenic bacteria in food. Methods: We designed six primers based on the gene coding sequences of inv A, specific to Salmonella. Each reaction solution contained 6.0 mM MgSO4, 1 M betaine, 1.6 mM dNTPs, 160 U/mL Bst DNA polymerase, 0.2 μM of both external primers, 0.8 μM of both internal primers, and 0.2 μM of both loop primers. The reaction temperature was 65°C. Results: Our amplified products were separated by 2% agarose gel electrophoresis. The detection limit was 10 CFU per reaction. Conclusions: RT-LAMP exhibited the same accuracy as the GB assay in detecting Salmonella in foods. RT-LAMP was highly specific and sensitive; hence, it may serve as an effective tool in detecting Salmonella.

The HBV Core Protein and Core Particle Both Bind to the PPiase Par14 and Par17 to Enhance Their Stabilities and HBV Replication

We recently reported that the PPIase Par14 and Par17 encoded by PIN4 upregulate HBV replication in an HBx-dependent manner by binding to conserved arginine–proline (RP) motifs of HBx. HBV core protein (HBc) has a conserved 133RP134 motif; therefore, we investigated whether Par14/Par17 bind to HBc and/or core particles. Native agarose gel electrophoresis (NAGE) and immunoblotting and co-immunoprecipitation were used. Chromatin immunoprecipitation from HBV-infected HepG2-hNTCP-C9 cells was performed. NAGE and immunoblotting revealed that Par14/Par17 bound to core particles and co-immunoprecipitation revealed that Par14/Par17 interacted with core particle assembly-defective, and dimer-positive HBc-Y132A. Thus, core particles and HBc interact with Par14/Par17. Par14/Par17 interacted with the HBc 133RP134 motif possibly via substrate-binding E46/D74 and E71/D99 motifs. Although Par14/Par17 dissociated from core particles upon heat treatment, they were detected in 0.2 N NaOH-treated opened-up core particles, demonstrating that Par14/Par17 bind outside and inside core particles. Furthermore, these interactions enhanced the stabilities of HBc and core particles. Like HBc-Y132A, HBc-R133D and HBc-R133E were core particle assembly-defective and dimer-positive, demonstrating that a negatively charged residue at position 133 cannot be tolerated for particle assembly. Although positively charged R133 is solely important for Par14/17 interactions, the 133RP134 motif is important for efficient HBV replication. Chromatin immunoprecipitation from HBV-infected cells revealed that the S19 and E46/D74 residues of Par14 and S44 and E71/D99 residues of Par17 were involved in recruitment of 133RP134 motif-containing HBc into cccDNA. Our results demonstrate that interactions of HBc, Par14/Par17, and cccDNA in the nucleus and core particle–Par14/Par17 interactions in the cytoplasm are important for HBV replication.

Frequencies of CYP2B6∗4,∗5, and ∗6 Alleles within an Iranian Population (Mazandaran)

Background. The human CYP2B subfamily consists of one functional gene (CYP2B6) and one pseudogene (CYP2B7P). Cytochrome P450 2B6 (CYP2B6) is a highly polymorphic enzyme that shows marked interindividual and interethnic variations. Currently, 38 alleles have been described, and some of the allelic variants have been associated with low enzyme activity. The aim of this study was to investigate the frequencies of CYP2B6∗4, CYP2B6∗5, and CYP2B6∗6 alleles in the Mazani ethnic group among Iranian Population. Methods. The study was conducted in 289 unrelated healthy volunteers. DNA was extracted from peripheral blood and analyzed by the PCR-RFLP protocol. The PCR product was digested with restriction enzymes and then separated using agarose gel electrophoresis. Results. The frequency of CYP2B6∗4, CYP2B6∗5, and CYP2B6∗6 in this study was 34.60%, 7.26%, and 34.54%, respectively. Conclusion. The frequency of the CYP2B6∗4 allele in the Mazani ethnic group was much higher (34.60%) than other population. The frequency of CYP2B6∗6 (34.54%) also was higher than its frequency in other previously reported population. But the frequency of CYP2B6∗5 in this study was lower than expected. These results will be useful in understanding the ethnic diversity in Iranian population and offer a preliminary basis for more rational use of drugs that are substrates for CYP2B6 in this population.

Low-Cost Imaging of Fluorescent DNA in Agarose Gel Electrophoresis Using Raspberry Pi Cameras

Low-cost analytical solutions built around microcomputers like the Raspberry Pi help to facilitate laboratory investigations in resource limited venues. Here, three camera modules (V1.3 with and without filter, as well as NoIR) that work with this microcomputer were assessed for their suitability in imaging fluorescent DNA following agarose gel electrophoresis. Evaluation of their utility was based on signal-to-noise (SNR) and noise variance metrics that were developed. Experiments conducted with samples were subjected to Polymerase Chain Reaction (PCR), and the amplified products were separated using gel electrophoresis and stained with Midori green. Image analysis revealed the NoIR camera performed the best with SNR and noise variance values of 21.7 and 0.222 respectively. In experiments conducted using UV LED lighting to simulate ethidium bromide (EtBr) excitation, the NoIR and V1.3 with filter removed cameras showed comparable SNR values.

High sensitivity-low cost detection of SARS-CoV-2 by two steps end point RT-PCR with agarose gel electrophoresis visualization

More than one year since Coronavirus disease 2019 (COVID-19) pandemic outbreak, the gold standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection is still the RT-qPCR. This is a limitation to increase testing capacities, particularly at developing countries, as expensive reagents and equipment are required. We developed a two steps end point RT-PCR reaction with SARS-CoV-2 Nucleocapsid (N) gene and Ribonuclease P (RNase P) specific primers where viral amplicons were verified by agarose gel electrophoresis. We carried out a clinical performance and analytical sensitivity evaluation for this two-steps end point RT-PCR method with 242 nasopharyngeal samples using the CDC RT-qPCR protocol as a gold standard technique. With a specificity of 95.8%, a sensitivity of 95.1%, and a limit of detection of 20 viral RNA copies/uL, this two steps end point RT-PCR assay is an affordable and reliable method for SARS-CoV-2 detection. This protocol would allow to extend COVID-19 diagnosis to basic molecular biology laboratories with a potential positive impact in surveillance programs at developing countries.

Alkaline Agarose Gel Electrophoresis

Alkaline agarose gels are run at high pH, which causes each thymine and guanine residue to lose a proton and thus prevents the formation of hydrogen bonds with their adenine and cytosine partners. The denatured DNA is maintained in a single-stranded state and migrates through an alkaline agarose gel as a function of its size. Other denaturants such as formamide and urea do not work well because they cause the agarose to become rubbery.

A Novel Lateral Flow Assay for Rapid and Sensitive Nucleic Acid Detection of Avibacterium paragallinarum

Avibacterium paragallinarum, the pathogen of infectious coryza, caused a highly contagious respiratory disease that poses a serious threat to chickens. Hence, it is necessary to do diagnostic screening for Av. paragallinarum. Existing technologies have been used for Av. paragallinarum testing, which, however, have some drawbacks such as time consuming and expensive that require well-trained personnel and sophisticated infrastructure, especially when they are limitedly feasible in some places for lack of resources. Nucleic acid hybridization-based lateral flow assay (LFA) is capable of dealing with these drawbacks, which is attributed to the advantages, such low cost, rapid, and simple. However, nucleic acid determination of Av. paragallinarum through LFA method has not been reported so far. In this study, we developed a novel LFA method that employed gold nanoparticle probes to detect amplified Av. paragallinarum dsDNA. Compared with agarose gel electrophoresis, this LFA strip was inexpensive, simple- to- use, and time- saving, which displayed the visual results within 5–8 min. This LFA strip had higher sensitivity that achieved the detection limit of 101 CFU/ml compared with 102 CFU/ml in agarose gel electrophoresis. Besides, great sensitivity was also shown in the LFA strip, and no cross reaction existed for other bacteria. Furthermore, Av. paragallinarum in clinical chickens with infectious coryza were perfectly detected by our established LFA strip. Our study is the first to develop the LFA integrated with amplification and sample preparation techniques for better nucleic acid detection of Av. paragallinarum, which holds great potential for rapid, accurate, and on-site determination methods for early diagnosis of Av. paragallinarum to control further spreading.

Sexing as a tool for reinsertion of Amazona aestiva parrots to nature: use of less invasive technique

The sexing birds is considered an important tool for behavioral studies and programs for the reintroduction of animals into the wild. Several techniques are used for this purpose, such as laparoscopy, magnetic resonance and molecular sexing. The first are considered more invasive and stressful for the animal, and the last is considered the most accurate. According to it, the aim of this study was to compare the effectiveness of using three sets of primers in the molecular sexing process of true parrots (Amazona aestiva). Blood samples from 10 animals were collected at a Wildlife Screening Center (CETAS) in Bahia, Brazil. The DNA was extracted and the molecular markers amplified by Polymerase Chain Reaction (PCR) using primer pairs P2/P8, 1237L/1272H and 2250F/2718R. The amplified material was visualized with electrophoresis performed at 2% agarose and 12.5% polyacrylamide gels. Among the primer sets used, the 2250F/2718R pair showed the best results for the sexing process, including visualization of the amplified products on an agarose gel. Agarose gel electrophoresis is considered to be faster and cheaper. The results revealed a sample composition of 5 males (0.5) and 5 females (0.5).