Impact of sample pooling strategy on 2019-nCoV RNA detection results / 中华检验医学杂志

Objective:To evaluate the impact of sample pooling strategy on 2019-nCoV RNA detection results.Methods:Ten negative swabs were stored in 6 ml virus transport medium, mixed thoroughly and diluted 1∶2 and 1∶10. Inactivated 2019-nCoV culture medium was added to simulate pooling samples: 10 pooling samples, 5 pooling samples and 1 swab sample. Extraction and amplification were made using three nucleic acid extraction reagents a, b, and c with different extraction methods and systems, as well as five 2019-nCoV detection reagents A-E with various template loading volumes and sensitivities respectively.Results:For the same sample, the Ct values of extracted templates a were 2.10±0.47 and 3.46±0.62 earlier than extracted templates b and c. For samples with identical amplifying, the Ct valves of N and ORF1ab gene of A reagent were 1.16±0.48 and 2.36±0.54 earlier than that of reagent B. Adding nucleic acid of 10 negative swabs to the amplification system lagged the Ct values of reagent A by about 1.36±0.32 Ct, while Ct values of reagent B were not affected. Extracted by regent a, a lag of 1.66±0.39 Ct on average was observed in C, D, and E reagents in detecting pooling samples of ten swabs as compared with one swab sample. When extracting 400 copies/ml pooling samples of ten swabs by reagent a, N gene could be detected by reagents C and E, but not by reagent D.Conclusion:Large amount of extraneous DNA is introduced by sample pooling, which could interfere the effiency of extraction and amplification. Strategies of using extraction reagents with large loading volume and high effiency, together with amplification reagents with large template volume and low limit of detection are helpful for ensuring detection sensitivity of pooling samples, and greatly reducing the risk of false negative results.

Evaluation of the Cutoff of Anti-HCV Antibody Enzyme-Linked Immunosorbent Assay in 7 Blood Station Laboratories / 中国实验血液学杂志

OBJECTIVE@#To evaluate the necessity and suitability of the anti-HCV ELISA teot gray zone setted up by 7 blood station laboratories.@*METHODS@#7 blood station laboratories were coded as 1, 2, 3, 4, 5, 6 and 7 respectively; 8 kinds of ELISA reagents were coded as A, B, C, D, E, F, G and H respectively. 1 or 2 of 8 ELISA reagents produced by different manufactories were used to detect the anti-HCV in specimens of same group by 7 blood station laboratories; the Westen blot was used to detect the specimens with difference of detected results so as to difine the serological status of specimens. The true positive rate of specimens detected by laboratories and gray zone-comfirined positive rate of specimens were accounted so as to analyze the necessity of setting up the gray zone for anti-HCV ELISA test of 7 blood station laboratories; the optimal cut-off value for anti-HCV ELISA test was determined in 7 blood station laborafories by ROC curve and the changes of sensitivity and specificity of 3 different cut-off value(laboratory work cut-off value, manifactory-recommended cun-off value and optimal cut-off value) were compared so as to analyze the suitability of gray zone for anti-HCV ELISA test in 7 blood station laboratories.@*RESULTS@#The true positive rate detected by 7 blood station laboratories, out of which coded 1 laboratory used 2 kinds of coded A, B reagents was 95.40%(1A), 99.23% (1B), 94.25% (2C), 96.17% (3D), 98.08% (4E), 96.93% (5F), 97.32%(6G) and 93.10%(7H). Except for 2C(94.25%) and 7H(93.10%), the true positive rate detected by laboratoies which not sutted up gray zone, the gray zone-con-firmed positive rate in 6 blood station laboratories setted up gray zone: was 0.00%, 0.00%, 21.43%, 0.00%, 0.00%, 0.00% and 38.89%. The comparison of 3 different cut-off valuces by ROC curve showed that the anti-HCV cut-off values in 5 laboratories(1B, 2C, 4E, 5F and 6G) were as follows: optimal cut-off value＞manufactory recommeded cut-off value＞laboratory work cut-off value, thus use of manufactory-recommeded cut-off value abreadly has reached the high sensitivity requinements for laboratory screening; however, the optimal cut-off value in laboratories 1A, 3B and 7H, thas the appropriate gray zone should be used. In 6 laboratories setting up gray zone, the gensitivity in 3D, 7H laboratories only a little improved (1.60% and 2.70% raspectively) in Eamparison between laboratory work cut-off value and manufactorg-recommeded cut-off value; moreover, the sensitivity in other laboratories not is changed, but the specificity decreased (0.20%-0.50%).@*CONCLUSION@#In addition to setting up the appropriate gray zone in laboratories 1A, 3D and 5H, the gray zone in other laboratories may be cancelled. Even in the same laboratory, the setting up the gray zone also should be scientifically assessed, the same scale cannot be blindly used, thus appropniate strategies should be established.

Establish and confirm of blood donor deferral criterion in HBsAg ELISA test / 国际检验医学杂志

Objective To establish and confirm the hepatitis B surface antigen(HBsAg) enzyme linked immunosorbent assay(ELISA) high specificity S/CO limit as blood donor deferral criterion.Methods A total of 783 HBsAg ELISA reactive and 588 non-reactive samples were collected, and confirmed by HBsAg electrochemiluminescence detection and neutralization test.Receiver operating characteristic curve (ROC curve) was used to evaluate the S/CO limit under 95% and 99% specificity.Another 124 HBsAg ELSIA reactive samples were tested for five kinds of hepatitis B virus(HBV) markers by using electrochemiluminescence detection to verify the blood donor deferral limit.The blood donor deferral limits of 3 laboratories, using the same reagents, were compared.Results The 95% specificity S/CO limit of two reagents were 0.24 and 0.65, the 99% specificity S/CO limit of two reagents were 3.89 and 3.62.The 99% specificity S/CO limit was set as the blood donor deferral criterion.Verify test indicated that the samples, with S/CO higher than the blood donor reentry limit of reagent 1 and 2, were all from HBV infected donor.The 99% specificity S/CO limits of reagent 1 in the other three laboratories were 3.77, 3.60 and 13.42 respectively.And the 99% specificity S/CO limits of reagent 2 in the other three laboratories were 27.73, 31.75 and 1.17.Conclusion The blood donor deferral limit of HBsAg ELISA could identify the true positive blood donor, and reduce the number of blood donor, entering the reentry process.It might not suit to adopt a unified donor deferral limit in different laboratories, even using the same reagents.

Identification of human cytomegalovirus phosphoprotein 65 in C57BL/6 and BXSB mice as a potential trigger of systemic lupus erythematosus related serum markers

Objective: To investigate the potential role of human cytomegalovirus lower matrix phosphoprotein 65 (HCMV-pp65) in murine systemic lupus erythematosus (SLE). Methods: The prokaryotic plasmid pET-28b-pp65 was constructed to express the HCMVpp65 protein. BXSB mice and C57BL/6 mice were inoculated with pp65 eukaryotic plasmid pcDNA3.0-pp65 intramuscularly 5 times at 2-week intervals, and then the blood of the mice was subsequently collected via the retro-orbital vein. Indirect ELISAs were used to evaluate the concentration of anti-pp65 immunoglobulin G, anti-double-stranded DNA and antinuclear antibodies. Interleukin-1β and tumor necrosis factor-α were also determined by competitive ELISA. At the same time, 3 major SLE-related circulating microRNAs were examined by quantitative RT-PCR. Results: The early production of autoantibodies was observed in pp65-immunized male BXSB as well as C57BL/6 mice. Overexpression of interleukin-1β and tumor necrosis factor-α were detected in pp65-immunized male BXSB mice. Quantitative RT-PCR analyses showed that three SLE related microRNAs (microRNA-126, microRNA-125a, and microRNA-146a) were downregulated in peripheral blood mononuclear cells of pp65-immunized mice. Conclusions: Our findings indicate that HCMV-pp65 immunization strongly triggers the development and progression of SLE-like disease in both BXSB and C57BL/6 mice, which indicates that the immune responses induced by HCMV-pp65 may be involved in the development of SLE.

External quality assessment on detection of hepatitis C virus RNA in clinical laboratories of China / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>As with many studies carried out in European countries, a quality assurance program has been established by the National Center for Clinical Laboratories in China (NCCL). The results showed that the external quality assessment significantly improves laboratory performance for quantitative evaluation of hepatitis C virus (HCV) RNA.</p><p><b>METHODS</b>Serum panels were delivered twice annually to the clinical laboratories which performed HCV RNA detection in China. Each panel made up of 5 coded samples. All laboratories were requested to carry out the detection within the required time period and report on testing results which contained qualitative and/or quantitative test findings, reagents used and relevant information about apparatus. All the positive samples were calibrated against the first International Standard for HCV RNA in a collaborative study and the range of comparison target value (TG) designated as +/- 0.5 log.</p><p><b>RESULTS</b>The numbers of laboratories reporting on qualitative testing results for the first and second time external quality assessment were 168 and 167 in the year of 2003 and increased to 209 and 233 in 2007; the numbers of laboratories reporting on quantitative testing results were 134 and 147 in 2003 and rose to 340 and 339 in 2007. Deviation between the mean value for quantitative results at home in 2003 and the target value was above 0.5 log, which was comparatively high. By 2007, the target value was close to the national average except for the low concentrated specimens (10(3) IU/ml). The percentage of results within the range of GM +/- 0.5 log(10) varied from 8.2% to 93.5%. Some laboratories had some difficulties in the exact quantification of the lowest (3.00 log IU/ml) as well as of the highest viral levels (6.37 log IU/ml) values, very near to the limits of the dynamic range of the assays.</p><p><b>CONCLUSIONS</b>The comparison of these results with the previous study confirms that a regular participation in external quality assessment (EQA) assures the achievement of a high proficiency level in the diagnosis of HCV infection. During the 5-year external quality assessment, sensitivity and accuracy of detection in most of the clinical laboratories have been evidently improved and the quality of kits has also been substantially improved.</p>

Establishment of the first national standards for nucleic acid amplification technology assay for HBV DNA / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To establish a Chinese national standard for a nucleic acid test (NAT) for HBV DNA.</p><p><b>METHODS</b>The candidate sample of HBV DNA positive plasma was diluted with HBV-negative human plasma. The sample was lyophilised with a concentration of approximately 300,000 copies/ml. The measurement methods used included Roche Amplicor assay (version 2.0) and real-time PCR. The lyophilised preparation was calibrated by the international standard (NIBSC code: 97/746) from NIBSC.</p><p><b>RESULTS</b>The quantity of this lyophilised preparation was (1.29+/-0.24) x 10(5)IU/ml in comparison with the international standard for HBV DNA 97/746. The stability test indicated that the sample was stable at room temperature (20 to 25 degrees C) for 2 weeks and at 37 degrees C for at least 1 week. Long-term stability was observed at 2 to 8 degrees C for 6 months and at -20 degrees C for more than 2 years with no significant changes. The vial-to-vial imprecision rate was 3.53%.</p><p><b>CONCLUSION</b>Based on the results of this study, our lyophilized sample can be used as a standard in China for the nucleic acid test (NAT) for HBV DNA.</p>

Lyophilized standards for the calibration of real time PCR assay for hepatitis C virus RNA / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Since October 1997, an international standard for hepatitis C virus (HCV) nucleic acid amplification technology assay, 96/790, has been available. We compared a series of lyophilized standards with known HCV RNA concentrations against the international standard in fluorescence quantitative PCR detection.</p><p><b>METHODS</b>A series of lyophilized sera were calibrated by ROCHE COBAS AMPLICOR HCV Monitor test against the international standard and sent to various manufacturers to analyse the samples using their own kits. Then calibration curves from the series were compared with that obtained from the external standard calibration curve with the manufacture's series.</p><p><b>RESULTS</b>The standard calibration curve with the series of lyophilized serum showed an excellent correlation (R(2) > 0.98), slope and intercept that were similar to those from the manufacture's series. When the standard calibration curve from the series of lyophilized standards were used to define the values of the given sample, lower coefficients of variation between kits from different manufactures were obtained.</p><p><b>CONCLUSION</b>The results showed that the lyophilized standards could be used to setup the standard calibration curve for clinical HCV RNA quantitative PCR detection.</p>

Selecting methods of controls concentration for internal quality control and continuity of control chart between different reagent lots for HBsAg qualitative detection / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To establish a model for one choosing controls with a suitable concentration for internal quality control (IQC) with qualitative ELISA detection, and a consecutive plotting method on Levey-Jennings control chart when reagent kit lot is changed.</p><p><b>METHODS</b>First, a series of control serum with 0.2, 0.5, 1.0, 2.0 and 5.0ng/ml HBsAg respectively were assessed for within-run and between-run precision according to NCCLs EP5 document. Then, a linear regression equation (y=bx + a) with best correlation coefficient (r > 0.99) was established based on S/CO values of the series of control serum. Finally, one could choose controls with S/CO value calculated from the equation (y = bx + a) minus the product of the S/CO value multiplying three-fold between-run CV to be still more than 1.0 for IQC use. For consecutive plotting on Levey-Jennings control chart when ELISA kit lot was changed, the new lot kits were used to detect the same series of HBsAg control serum as above. Then, a new linear regression equation (y2 = b2x2 + a2) with best correlation coefficient was obtained. The old one (y1 =b1x1 + a1) could be obtained based on the mean values from above precision assessment. The S/CO value of a control serum detected by new lot kit could be changed to that detected by old kit lot based on the factor of y2/y1. Therefore, the plotting on primary Levey-Jennings control chart could be continued.</p><p><b>RESULTS</b>The within-run coefficient of variation CV of the ELISA method for control serum with 0.2, 0.5, 1.0, 2.0 and 5.0ng/ml HBsAg were 11.08%, 9.49%, 9.83%, 9.18% and 7.25%, respectively, and between-run CV were 13.25%, 14.03%, 15.11%, 13.29% and 9.92%. The linear regression equation with best correlation coefficient from a test at random was y = 3.509x + 0.180. The suitable concentration of control serum for IQC could be 0.5ng/ml or 1.0ng/ml. The linear regression equation from the old lot and other two new lots of the ELISA kits were y1 = 3.550(x1) + 0.226, y2 = 3.238(x2) +0.388, and y3 =3.428(x3) + 0.148, respectively. Then, the transferring factors of 0.960 (y2/y1) and 0.908 (y3/y1) were obtained.</p><p><b>CONCLUSION</b>The results shows that the model established for IQC control serum concentration selecting and for consecutive plotting on control chart when the reagent lot is changed is effective and practical.</p>

Construction and expression of RNase-resisting virus-like particles containing PSA mRNA / 中华老年医学杂志

Objective To construct an expression system to produce the virus-like particles containing a part of the sequence of PSA mRNA, which are ribonuclease-resistant due to the encapsulation of the mRNA by bacteriophage MS2 coat proteins. Methods The PCR products of PSA cDNA fragments were cloned to TA vector pBS-T, then the targeted segments could be obtained when the pBS-T-PSA were digested by restriction endonuclease Hind Ⅲ and cloned to prokaryocytic expression vector pNCCL1. The recombinant plasmids named PNCCL1-PSA were transfected into E. Coli BL21-DE3 and induced to express with IPTG. Results The recombinant plasmids were successfully constructed. The bacteriophage MS2 coat protein which expressed in BL21 can self- assemble to form ribonuclease resistant virus-like particles and the PSA mRNA was encapsulated into virus-like particles. Conclusions The virus-like particle containing PSA mRNA can be expressed in prokaryocyte and it can be used as standard and control in detecting PSA mRNA. It provides a new, stable and ribonuclease-resistant RNA standard in RNA detection.

Detection of anti-dsDNA antibody in SLE patients by enzyme marking staphylococcal protein A (SPA) / 中国免疫学杂志

The results of the detection of fhe antibody against dsDNA in 244 sera by immunohisto—chemical method of enzyme marking SPA were reported and compared with immunoflurescence assay and enzyme marking antibody method. Positive rate in 31 cases with SLE was 71%. Of the 31 cases 21 with SLE in theactive phase were all positive,1 out of 10 cases at the recovery stage was positive,2 outof 152 cases with other connective tissue and non connective tissue disease were weaklypositive,61 normal persons were all negative.The overall agreement was the same asthe immunofluorescence and enzyme marking antibody method.Enzyme marking SPAmethod offers a number of significant advantageous.This method was easily operated,did not need to prepare second antibody,and special equipment was not needed.It can beused clinically.