Soft interface confined DNA walker for sensitive and specific detection of SARS-CoV-2 variants.

Nucleic acid detection is conducive to preventing the spread of COVID-19 pandemic. In this work, we successfully designed a soft interface confined DNA walker by anchoring hairpin reporter probes on cell membranes for the detection of SARS-CoV-2 variants. In the presence of target RNA, the cyclic self-assembly reaction occurred between hairpin probes H1 and H2, and the continuous walking of target RNA on cell membranes led to the gradual amplification of fluorescence signal. The enrichment of H1 on membranes and the unique fluidity of membranes promoted the collision efficiency between DNA strands in the reaction process, endowing this method with high sensitivity. In addition, the double-blind test of synthetic RNA in 5% normal human serum demonstrated the good stability and anti-interference in complex environment of this method, which exhibited great potential in clinical diagnostics.

Diagnostic significance of alanine aminotransferase isoenzymes in alcoholic and non-alcoholic fatty liver cancers.

OBJECTIVES: Alanine aminotransferase (ALT) expression is highly elevated in the serum of patients with hepatocellular carcinoma. However, the role of ALT isoenzymes in the total ALT activity remains unclear. In the present study, we systematically investigated the role of ALT isoenzymes in alcoholic and non-alcoholic fatty liver cancer. MATERIALS AND METHODS: The expression of ALT1 and ALT2 at the mRNA and protein levels in 25 paired primary liver cancer tissues was detected by reverse transcription quantitative PCR (RT-qPCR), Western blotting, and immunohistochemistry. Serum ALT activity was determined using an automated biochemical analyzer. RESULTS: The mRNA and protein expression levels of ALT1 and ALT2 were lower in the tissues of alcoholic and non-alcoholic fatty liver cancers than in the paracancerous tissues. Notably, ALT2 was highly expressed in non-alcoholic fatty liver cancer tissues compared with alcoholic fatty liver cancer tissues. Total serum ALT activity was mainly contributed by ALT1 in alcoholic fatty liver cancer, whereas ALT1 contributed only marginally more to the total ALT activity than ALT2 in non-alcoholic fatty liver cancer. ALT2/ALT1 ratio can well discriminate normal control group, alcoholic liver cancer and non-alcoholic liver cancer. CONCLUSION: ALT1 contributed more to the total ALT activity than ALT2 in both alcoholic and non-alcoholic fatty liver cancer. Serum ALT2 to ALT activity was higher in non-alcoholic fatty liver cancer than that in alcoholic fatty liver cancer. ALT2/ALT1 ratio has some diagnostic significance for alcoholic and non-alcoholic liver cancer.

Novel cytosensor for accurate detection of circulating tumor cells based on a dual-recognition strategy and BSA@Ag@Ir metallic-organic nanoclusters.

Herein, based on a dual-recognition strategy and BSA@Ag@Ir metallic-organic nanoclusters (BSA@Ag@Ir MONs), a highly specific and sensitive cytosensor was developed for detecting circulating tumor cells (CTCs). To amplify current signal, novel BSA@Ag@Ir MONs with outstanding catalytic activity and huge specific surface area were synthesized, and conjugated with hairpin DNA strands as signal probes. Orion carbon black 40 (Ocb40)//AuNPs were firstly used to modify electrode to increase its conductivity and surface area. Moreover, the dual recognition strategy based on DNA proximity effect was designed to improve the specificity of cytosensor. When two capture probes respectively bound to two adjacent membrane markers of target cells, the probes could form the associative toehold through the proximity effect to capture the signal probes. Only CTCs simultaneously expressing two membrane markers could be captured and generate current responses. The developed cytosensor could detect CTCs in the range of 3 - 3 × 106 cells mL-1 with a detection limit of 1 cell mL-1. Notably, the cytosensor could accurately identify CTCs even in whole blood. Therefore, this cytosensor has great potential for application in biological science, biomedical engineering and personalized medicine.

The correlation between expression of sip protein in different serotypes of group b streptococcus and diagnosis.

Since the surface immunogenic protein (Sip) of group B streptococcus was identified, it's immunogenicity and its potential as a universal vaccine candidate has been evaluated extensively. We developed recombinant Sip protein and used it for monoclonal antibody generation to develop immunochromatographic test kit for GBS detection. The test of bacteria and culture media revealed the correlation between Sip protein expression and diagnosis discrepancy, which has never been reported. Furthermore, not only the surface accessibility of the Sip protein may vary from strains or serotypes; the secretion level of Sip protein may also vary.

Development of an immunochromatographic lateral flow device for rapid diagnosis of Vibrio cholerae O1 serotype Ogawa.

OBJECTIVES: Cholera is an acute malignant infectious disease caused by the bacteria Vibrio cholerae leading to severe dehydrating diarrhea and vomiting, even high rates of mortality in some cases. However, the prevention of the epidemic disease is achievable if proper sanitation practices are followed, provided the accurate and prompt diagnosis of each prevalent serotype in cholera epidemic. The current gold standard of bacterial culture is inadequate for rapid diagnosis. Our aim is to develop an immunochromatographic test format for O1 serotype Ogawa diagnosis and provide the need for better epidemic prevention and early response. DESIGN AND METHODS: The monoclonal antibodies were raised in conventional method and subsequently screened for a match pair. A variety of related and unrelated bacteria strains recruited were employed to test their sensitivity, specificity etc. by indirect ELISA. The human fecal samples were used to test the final lateral-flow device product to satisfy the measurement requirement. RESULTS: A new monoclonal antibody (McAb) pair, named IXiao3G6 and IXiao1D9, was generated, which is specifically against V. cholerae O1 serotype Ogawa. Additionally, we developed an immunochromatographic lateral flow device (LFD) using this McAb pair for the highly specific and rapid (5 min) detection of Ogawa. CONCLUSIONS: Our product has advantages of simplicity and precision, and can benefit the scene and elementary medical institutions.

Development of an immunoassay for differentiating human immunodeficiency virus infections--from vaccine-induced immune response in Tiantan vaccine trials in China.

OBJECTIVE: To develop a Rapid Flow-through assay for distinguishing replicating Tiantan vaccine-generated serological response from true HIV infection. DESIGN AND METHOD: A Rapid Flow-through Test including gp41, gp36, sk1, sk2 and sk3 antigens was established and the performance of the assay was evaluated in clinical studies and compared with ELISA assay. RESULTS: Sk1, sk2 and sk3 peptides performed at 100% specificity and slightly but not significantly different sensitivities between ELISA assay (92%, 76% and 41%) and Flow-through Test Kit (92%, 75% and 40%) in diagnosing HIV-1 infections. Of particular importance, Tiantan vaccine recipients that gave false-positive results in gp41 serodetection scored negative for sk1, sk2 and sk3 antibodies. CONCLUSION: The Rapid Flow-through Test could be a robust tool in both diagnosing HIV-1/2 infections and differentiating between vaccines induced immunity and immunity resulting from natural exposure, thus serving as potential implementation in HIV vaccine trials.

Phenotypic assay of a hepatitis B virus strain carrying an rtS246T variant using a new strategy.

Phenotypic assays of hepatitis B virus (HBV) play an important role in research related to the problem of drug resistance that emerges during long-term nucleot(s)ide therapy in patients with chronic hepatitis B. Most of the phenotypic assay systems that are available currently rely on the transfection of recombinant replication-competent HBV DNA into hepatoma cell lines. Cloning clinical HBV isolates using conventional digestion-and-ligation techniques to generate replication-competent recombinants can be very difficult because of the sequence heterogeneity and unique structure of the HBV genome. In this study, a new strategy for constructing an HBV 1.1× recombinant was developed. The core of this strategy is the "fragment substitution reaction" (FSR). FSR allows PCR fragments to be cloned without digestion or ligation, providing a new tool for cloning fragments or genomes amplified from serum HBV DNA, and therefore making the assay of HBV phenotypes more convenient. Using this strategy, a phenotypic assay was performed on an HBV strain carrying an rtS246T variant isolated from a patient with chronic hepatitis B that was only responsive partially to entecavir therapy. The results indicated that this strain is sensitive to entecavir in vitro.