CRISPR/Cas13a-based single-nucleotide polymorphism detection for reliable determination of ABO blood group genotypes.

The ABO blood group plays an important role in blood transfusion, linkage analysis, individual identification, etc. Serologic methods of blood typing are gold standards for the time being, which require stable typing antisera and fresh blood samples and are labor intensive. At present, reliable determination of ABO blood group genotypes based on single-nucleotide polymorphisms (SNPs) among A, B, and O alleles remains necessary. Thus, in this work, CRISPR/Cas13a-mediated genotyping for the ABO blood group by detecting SNPs between different alleles was proposed. The ABO*O.01.01(c.261delG) allele (G for the A/B allele and del for the O allele) and ABO*B.01(c.796C > A) allele (C for the A/O allele and A for the B allele) were selected to determine the six genotypes (AA, AO, BB, BO, OO, and AB) of the ABO blood group. Multiplex PCR was adapted to simultaneously amplify the two loci. CRISPR/Cas13a was then used to specifically differentiate ABO*O.01.01(c.261delG) and ABO*B.01(c.796C > A) of A, B, and O alleles. Highly accurate determination of different genotypes was achieved with a limit of detection of 50 pg per reaction within 60 min. The reliability of this method was further validated based on its applicability in detecting buccal swab samples with six genotypes. The results were compared with those of serological and sequencing methods, with 100% accuracy. Thus, the CRISPR/Cas13a-mediated assay shows great application potential in the reliable identification of ABO blood group genotypes in a wide range of samples, eliminating the need to collect fresh blood samples in the traditional method.

Correlation analysis of hepatitis C virus core antigen and low viral loads: Can core antigen replace nucleic acid test?

Value of hepatitis C virus (HCV) core antigen (cAg) test has been controversy in patients with low HCV loads for its lower sensitivity. We assessed correlation between HCV-cAg and HCV RNA in serum samples with low viral loads and analyzed the performance of HCV-cAg assay in determining diagnosis and treatment outcomes in chronic hepatitis C patients. Both HCV RNA and HCV-cAg were detected for 2298 serum samples. Correlation analysis was performed between the two tests. Receiver operating characteristics (ROC) curve was used to assess value of HCV-cAg test in determining diagnosis and response outcomes at the different HCV RNA thresholds. The two tests were correlated very well, and moreover, correlation in the low viral load group was higher than that in the high viral load group (r value: 0.901 and 0.517). Positive agreement of HCV-cAg ≥ 3 fmol/L was as high as 97.0% for HCV RNA ≥ 1000 IU/mL, and its negative agreement for HCV RNA < 15 IU/mL was up to 98.9% in all samples. Area under ROCs ranged from 0.939 to 0.992, regardless of HCV RNA thresholds. When lower limit of detection of HCV RNA was 15, 100 or 1000 IU/mL, positive predictive value of HCV-cAg was 96.8%, 98.8% or 92.4%, and its negative predictive value was 87.0%, 89.9% or 98.3%, respectively, on the basis of different cutoff values. High-sensitivity HCV-cAg detection may likely replace HCV RNA to confirm the existence of HCV and to guide the treatment of chronic HCV infection.

Direct antiviral agents upregulate natural killer cell potential activity in chronic hepatitis C patients.

Direct antiviral agents (DAAs) can eliminate hepatitis C virus rapidly and make chronic hepatitis C (CHC) curable. The changes in the innate immune system during treatment with DAAs are still in dispute. To investigate how the functions of natural killer (NK) cells change during and after treatment with DAAs in each NK cell subset. Thirteen CHC patients were treated with sofosbuvir/ledipasvir, and the expression levels of NKp46 and NKG2A were tested via flow cytometry at baseline, at 2, 4, 8 and 12 weeks during the therapy and 12 and 24 weeks after the end of treatment; expression levels were compared between CHC patients and 13 healthy controls. A redirected killing assay was used to detect the cytotoxicity of NK cells. After coculturing NK cells with JFH-Huh7 cells for 72 h, HCV RNA was tested to analyze the inhibition ability of NK cells. All patients achieved sustained virologic response. The expression of the activating receptor NKp46 was decreased first at week 8 during therapy with DAAs and then increased and normalized to levels in healthy controls after treatment with DAAs. The expression of the inhibitory receptor NKG2A was decreased during and after treatment with DAAs. Each NK cell subset has a similar changing trend during and after treatment with DAAs, although some differences can be found earlier and later. The ratio of NKp46 and NKG2A was upregulated after treatment with DAAs. CD56bright NK cells have less amplitude in the frequency ratio changes after treatment with DAAs. The coculture results showed that both the specific lysis and the inhibition of HCV replication were significantly upregulated after treatment with DAAs. DAA treatments can affect patients' NK cell function. After DAA treatments, the expression of functional markers is downregulated, but the potential activity of NK cells is upregulated. The function of NK cells is normalized to levels in healthy controls. CD56bright NK cells play an important role in this process.

Recovery of natural killer cells is mainly in post-treatment period in chronic hepatitis C patients treated with sofosbuvir plus ledipasvir.

AIM: To investigate how natural killer (NK) cells are affected in the elimination of hepatitis C virus (HCV) by sofosbuvir/ledipasvir, two highly effective direct-acting antivirals (DAAs). METHODS: Thirteen treatment-naïve and treatment-experienced chronic hepatitis C (CHC) patients were treated with sofosbuvir/ledipasvir, and NK cells were detected at baseline, weeks 2, 4, 8 and 12 during therapy, and week post of treatment (Pt)-12 and 24 after the end of therapy by multicolor flow cytometry and compared with those from 13 healthy controls. RESULTS: All patients achieved sustained virological response. There was a significant decline in CD56bright NK cell frequencies at week 8 (P = 0.002) and week 12 (P = 0.003), which were altered to the level comparable to healthy controls at week Pt-12, but no difference was observed in the frequency of CD56dim NK cells. Compared with healthy controls, the expression levels of NKG2A, NKp30 and CD94 on NK cells from CHC patients at baseline were higher. NKG2A, NKp30 and CD94 started to recover at week 12 and reached the levels similar to those of healthy controls at week Pt-12 or Pt-24. Before treatment, patients have higher interferon (IFN)-γ and perforin levels than healthy controls, and IFN-γ started to recover at week 8 and reached the normalized level at week Pt-12. CONCLUSION: NK cells of CHC patients can be affected by DAAs, and phenotypes and function of NK cells recover not at early stage but mainly after the end of sofosbuvir/ledipasvir treatment.

Optimization, purification and structural characterization of a dextran produced by L. mesenteroides isolated from Chinese sauerkraut.

A higher yield of dextran strain Leuconstoc mesenteroides TDS2-19 was isolated from Chinese sauerkraut juice. Effects of the three main factors on exopolysaccharide (EPS) yield were investigated by central composite design (CCD) and the optimum composition was sucrose 117.48g/L, sodium acetate 4.10g/L, and initial pH 6.88. Optimum results showed that EPS yield was increased to 71.23±2.25g/L in 48h fermentation, 31.24% higher than before. The molecular weight (Mw) of ESP was 8.79×107Da, as determined by high-performance size-exclusion chromatography (HPSEC). Fourier transform infrared spectra (FT-IR) and nuclear magnetic resonance spectra (NMR) showed that the polysaccharide synthesized by L. mesenteroides TDS2-19 in the MRS medium was dextran with a peak, a linear backbone composed of consecutive α-(1→6)-linked d-glucopyranose units. No branching was observed in the dextran structure. The present study suggested that L. mesenteroides TDS2-19 might be used for the industrial-scale production of linear dextran.