Diagnostic Kits For Detection Research Articles

Plant Molecular Farming as a Strategy Against COVID-19 - The Italian Perspective.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed more than 37,000 people in Italy and has caused widespread socioeconomic disruption. Urgent measures are needed to contain and control the virus, particularly diagnostic kits for detection and surveillance, therapeutics to reduce mortality among the severely affected, and vaccines to protect the remaining population. Here we discuss the potential role of plant molecular farming in the rapid and scalable supply of protein antigens as reagents and vaccine candidates, antibodies for virus detection and passive immunotherapy, other therapeutic proteins, and virus-like particles as novel vaccine platforms. We calculate the amount of infrastructure and production capacity needed to deal with predictable subsequent waves of COVID-19 in Italy by pooling expertise in plant molecular farming, epidemiology and the Italian health system. We calculate the investment required in molecular farming infrastructure that would enable us to capitalize on this technology, and provide a roadmap for the development of diagnostic reagents and biopharmaceuticals using molecular farming in plants to complement production methods based on the cultivation of microbes and mammalian cells.

A sensitive gold-nanorods-based nanobiosensor for specific detection of Campylobacter jejuni and Campylobacter coli

BackgroundCampylobacteriosis is a zoonotic infectious disease that can be mostly undiagnosed or unreported due to fastidious Campylobacter species. The aim of this study was to develop a simple, sensitive, and quick assay for the detection of Campylobacter spp. and taking advantage of the great sensitivity of gold nanorods (GNRs) to trace changes in the local environment and interparticle distance.MethodsCharacterized GNRs were modified by specific ssDNA probes of cadF gene. First, the biosensor was evaluated using recombinant plasmid (pTG19-T/cadF) and synthetic single-stranded 95 bp gene, followed by a collection of the extracted DNAs of the stool samples. The sensing strategy was compared by culture, PCR, and real-time PCR.Results and discussionAnalysis of 283 specimens showed successful detection of Campylobacter spp. in 44 cases (16%), which was comparable to culture (7%), PCR (15%), and real-time PCR (18%). In comparison with real-time PCR, the sensitivity of the biosensor was reported 88%, while the specificity test for all assays was the same (100%). However, it was not able to detect Campylobacter in 6 positive clinical samples, as compared to real-time PCR. The limit of detection was calculated to be the same for the biosensor and real-time PCR (102 copy number/mL).ConclusionsTaking high speed and simplicity of this assay into consideration, the plasmonic nanobiosensor could pave the way in designing a new generation of diagnostic kits for detection of C. jejuni and C. coli species in clinical laboratories.

A Comparison of Diagnostics Kits Used in Russia for Identification of Antigens of Noroviruses

Three diagnostic kits for detection of antigens of noroviruses were evaluated and compared. These were (1) the RIDAQUICK® Norovirus test kit (R-Biopharm AG, Germany), (2) the RIDASCREEN® Norovirus test kit (R-Biopharm AG, Germany), and (3) the Norovirus-antigen IFA-BEST kit (VECTOR-BEST, Russia). The study was carried out with a panel containing 90 norovirus-positive (GI/GII) stool samples (GI.2, GI.3, GI.4, GI.6, GII.1, GII.2, GII.3 and GII.4 (including GII.4_New_Orlean and GII.4_Sydney), and GII.5, GII.6, GII.7, GII.10, GII.15. and GII.17) and 30 negative samples. The AmpliSens OKI scrin kit (FSR no. 2008/02265 dated November 17, 2011) was used as for comparison for identification of noroviruses of the GII genogroup. A technique that included confirmative sequencing of the capsid and polymerase gene fragments was used to determine noroviruses of the GI genogroup. Only 26 of 90 (28.9%) reference-positive samples were determined as norovirus-positive ones with any of the three diagnostic kits used in the present study. False-positive results of the test were revealed only with the Norovirus-antigen IFA-BEST kit in 8 out of 30 (26.7%) reference-negative samples. The estimated indices of diagnostic sensitivity and specificity of the RIDAQUICK® Norovirus test kit were 12.2 and 100%, respectively. They were 26.7 and 100% for the RIDASCREEN® Norovirus test kit and 17.8 and 73.3% for the Norovirus-antigen IFA-BEST kit.

Real-Time PCR: Analyte-Specific Reagents versus FDA-Approved Kits

In the January 2006 issue of the Clinical Microbiology Reviews, Espy et al. published a detailed comprehensive review on real-time PCR in clinical microbiology (M. J. Espy, J. R. Uhl, L. M. Sloan, S. P. Buckwalter, M. F. Jones, E. A. Vetter, J. D. C. Yao, N. L. Wengenack, J. E. Rosenblatt, F. R. Cockerill, and T. F. Smith, Clin. Microbiol. Rev. 19:165–256, 2006). This new technology is revolutionizing laboratory diagnosis of human pathogens. The authors covered extensively the literature on real-time PCR as well as the wide array of commercially available analyte-specific reagents (ASR) and products for research use only for real-time PCR but did not provide adequate coverage of available rapid real-time PCR diagnostic kits for detection of bacterial pathogens that are approved by the Food and Drug Administration (FDA). Currently, there are two FDA-approved real-time PCR kits that can replace standard culture (H. D. Davies, M. A. Miller, S. Faro, D. Gregson, S. C. Kehl, and J. A. Jordan, Clin. Infect. Dis. 39: 1129–1135, 2004; D. K. Warren, R. S. Liao, L. R. Merz, M. Eveland, and W. M. Dunne, J. Clin. Microbiol. 42:5578– 5581, 2004) and which are both commercialized by GeneOhm Sciences (a BD Company). The first, IDI-Strep BTM, was approved by the FDA in March 2003 for detection of group B streptococci from vaginal/anal swab specimens obtained from pregnant women during delivery (Davies et al., Clin. Infect. Dis. 39:1129–1135, 2004; F. J. Picard and M.G. Bergeron, Eur. J. Clin. Microbiol. Infect. Dis. 23:665–671, 2004). The second, IDIMRSATM, was approved in March 2004 for detection of methicillin-resistant Staphylococcus aureus from a nasal swab specimen (M. G. Bergeron, A. Huletsky, F. J. Picard, and M. Boissinot, Nature 430: 141, 2004; A. Huletsky, R. Giroux, V. Rossbach, M. Gagnon, M. Vaillancourt, M. Bernier, F. Gagnon, K. Truchon, M. Bastien, F. J. Picard, A. van Belkum, M. Ouellette, P. H. Roy, and M. G. Bergeron, J. Clin. Microbiol. 42:1875–1884; Warren et al., J. Clin. Microbiol. 42:5578– 5581, 2004).

VERIFIKASI cDNA T29 SEBAGAI KANDIDAT GEN PENGKODE PROTEIN Toxoplasma gondii DENGAN METODE SDS PAGE

The process of cDNA construction from mRNA isolated from Toxoplasma gondii has been done. There were 7 candidates cDNA which one of them is called T29. Since Toxoplasma gondii is the cause of toxoplasmosis infection, cloning the gene encoding protein from this parasite provides an important tool for developing diagnostic kit for detection of toxoplasmosis. Digestion of the cDNA T29 with EcoRI which is the restriction site where the cDNA was inserted yielded a 1.862 bp fragment. The fragment was subcloned into E. coli expression vector pMal-p2x and transformed into E.coli strain TB1. Colonies of TB1 were grown on ampicillin plates and the recombinant plasmid was extracted using the standard procedure. The plasmid was digested using EcoRI and PstI, checked by PCR amplification using malE and M13/pUC primers. The recombinant plasmid was expressed in TB1 and the protein extracted was ran in SDS PAGE to observe the presence of the expressed protein. Based on the data from this experiment, there was no expression result of the expressed cDNA which was confirm by the PCR result. Therefore, it was concluded that cDNA T29 was not carrying the gene coding for protein from parasite Toxoplasma gondii.

Cloning, expression and antigenic analysis of heat shock protein A gene of human Helicobacter pylori

AIM: To construct a recombinant vector containing gene encoding heat shock protein A with a Mr of 13 000 from human Helicobacter pylori (H pylori) and express it in E. coli BL21, and to explore the antigenicity. METHODS: The target gene was amplified from H pylori chromosome by PCR, and then inserted into the prokaryotic expression vector pET32a (+) digested by restrictive endonuclease enzymes of kpn , BamH I simultaneously. The recombinant vector was transformed and expressed in E.coli BL21.The antigenicity of recombinant fusion protein was analysed by Western blot. RESULTS: Enzyme digestion and sequencing analysis showed that the target gene has been inserted into the recombinant vector, but as compared with the gene reported by GenBank, 1.6 % of gene mutation and 1.6 % of amino acid residues change in H pylori occurred, respectively. SDS-PAGE analysis showed that the recombinant vector could be expressed in E.coli BL21, the relative molecular mass (Mr) of expressed product was 33×103, while Mr of protein expressed by pET32a (+) was about 20×103, and soluble fusion expression product accounted for 18.96 % of total bacterial protein. After purification with Ni+-NTA agarose resin, the purity of recombinant fusion protein was about 95 %. Western blot result showed that recombinant fusion protein could be recognized by anti-H pylori positive serum, suggesting that the protein had good antigenicity. CONCLUSION: The gene encoding H pylori heat shock protein A has been cloned and expressed successfully. The results lay the foundation for development of H pylori protein vaccine and a quick diagnostic kit for detection of H pylori infection. Jiang Z, Pu D, Huang AL, Tao XH, Wang PL. Cloning, expression and antigenic analysis of heat shock protein A gene of human Helicobacter pylori. Shijie Huaren Xiaohua Zazhi 2003;11(10):1480-1484