[Show abstract][Hide abstract] ABSTRACT: In recent years, nucleic acid tests for detection of measles virus RNA have been widely applied in laboratories belonging to the measles surveillance system of China. An external quality assessment program was established by the National Center for Clinical Laboratories to evaluate the performance of nucleic acid tests for measles virus. The external quality assessment panel, which consisted of 10 specimens, was prepared using armored RNAs, complex of noninfectious MS2 bacteriophage coat proteins encapsulated RNA of measles virus, as measles virus surrogate controls. Conserved sequences amplified from a circulating measles virus strain or from a vaccine strain were encapsulated into these armored RNAs. Forty-one participating laboratories from 15 provinces, municipalities, or autonomous regions that currently conduct molecular detection of measles virus enrolled in the external quality assessment program, including 40 measles surveillance system laboratories and one diagnostic reagent manufacturer. Forty laboratories used commercial reverse transcription-quantitative PCR kits, with only one laboratory applying a conventional PCR method developed in-house. The results indicated that most of the participants (38/41, 92.7%) were able to accurately detect the panel with 100% sensitivity and 100% specificity. Although a wide range of commercially available kits for nucleic acid extraction and reverse transcription polymerase chain reaction were used by the participants, only two false-negative results and one false-positive result were generated; these were generated by three separate laboratories. Both false-negative results were obtained with tests performed on specimens with the lowest concentration (1.2 × 104 genomic equivalents/mL). In addition, all 18 participants from Beijing achieved 100% sensitivity and 100% specificity. Overall, we conclude that the majority of the laboratories evaluated have reliable diagnostic capacities for the detection of measles virus.
PLoS ONE 08/2015; 10(8):e0134681. DOI:10.1371/journal.pone.0134681 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: With the rapid development of molecular diagnostic techniques, there is a growing need for quality controls and standards with favorable properties to monitor the entire detection process. In this study, we describe a novel method to produce armored hepatitis B virus (HBV) and human papillomavirus (HPV) DNA for use in nucleic acid tests, which was confirmed to be stable, homogeneous, noninfectious, nuclease resistant, and safe for shipping. We demonstrated that MS2 bacteriophage could successfully package double-stranded DNA of 1.3-, 3-, 3.5-, and 6.5-kb length into viral capsids with high reassembly efficiency. This is the first application of RNA bacteriophage MS2 as a platform to encapsulate double-stranded DNA, forming virus-like particles (VLPs) which were indistinguishable from native MS2 capsids in size and morphology. Moreover, by analyzing the interaction mechanism of pac site and the MS2 coat protein (CP), we found that in addition to the recognized initiation signal TR-RNA, TR-DNA can also trigger spontaneous reassembly of CP dimers, providing a more convenient and feasible method of assembly. In conclusion, this straightforward and reliable manufacturing approach makes armored DNA an ideal control and standard for use in clinical laboratory tests and diagnostics, possessing prospects for broad application, especially providing a new platform for the production of quality controls for DNA viruses.
Applied Microbiology and Biotechnology 05/2015; DOI:10.1007/s00253-015-6664-4 · 3.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previously, we developed a novel microRNA (miRNA) delivery system based on bacteriophage MS2 virus-like particles (MS2 VLPs). In this current study, we used this system to transport miR-146a into human peripheral blood mononuclear cells (PBMCs), and demonstrated the inhibition of osteoclastogenesis in precursors. Two cytokines, receptor activator of NF-κB ligand (RANKL), and macrophage-colony stimulating factor (M-CSF) were used to induce osteoclastogenesis. MS2 VLPs were transfected into PBMCs. qRT-PCR was applied to measure expression levels of miR-146a and osteoclast (OC)-specific genes. Western blot (WB) was conducted to evaluate miR-146a downstream target proteins: epidermal growth factor receptor (EGFR) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6). The formation and activity of OCs were assessed by cytochemical staining and bone resorption assay, respectively. In PBMCs treated with MS2-miR146a VLPs, qRT-PCR assays showed increased expression of miR-146a (p < 0.01) and decreased expression of all four OC-specific genes (p < 0.05). WB results indicated decreased expression of EGFR (p < 0.01) and TRAF6 (p < 0.05). The number of OCs decreased markedly and bone resorption assay demonstrated inhibited activity. This miR-146a delivery system could be applied to induce overexpression of miR-146a and to inhibit the differentiation and function of OCs.
International Journal of Molecular Sciences 04/2015; 16(4):8337-50. DOI:10.3390/ijms16048337 · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Warfarin is the most commonly used oral anticoagulant in clinical practice. The cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex 1 (VKORC1) genotypes have been confirmed to be associated with warfarin dose requirements. Accurate genotyping results are of particular importance for the obtaining of reliable genotype-guided warfarin dosing information. This study aims to determine analytic performance of laboratories offering CYP2C9 and VKORC1 testing in China. A proficiency panel of 15 validated cell samples covering common CYP2C9 and VKORC1 genetic polymorphisms was provided to 31 participating laboratories and their genotyping results were evaluated. Fourteen data sets (45.2%) performed well with the entire panel of samples, and 17 data sets (54.8%) reported at least one genotyping error. For VKORC1 (-1639G>A), participating laboratories were 100% successful in detecting genotypes of GG, GA and AA. For CYP2C9, participants were greater than 90% successful in detecting genotypes of *1/*1, *1/*2, *1/*3, *2/*3, and *3/*3. However, 15 laboratories failed to detect rarely encountered variant genotype *2/*2. The poor performance of CYP2C9 genotyping may be due to the limitation of methodologies used for detecting CYP2C9*2 allele. The proficiency testing (PT) survey highlighted the need for improving genotyping accuracy for some laboratories in this field.
Journal of cardiovascular pharmacology 03/2015; Publish Ahead of Print. DOI:10.1097/FJC.0000000000000254 · 2.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
The participation of laboratories in external quality assessment (EQA) programs is required for the quality assurance of nucleic acid amplification of Chlamydia trachomatis. This study aimed to construct a new quality control (QC) material applicated in EQA of C. trachomatis PCR.
A QC material-HTB-SiHa cells transfected with a recombinant plasmid containing the cryptic plasmid sequence-was constructed for C. trachomatis PCR detection, and four different panels, each consisting of 4 positive samples with serial dilution of the constructed QC material and 1 negative sample, were distributed by the National Center for Clinical Laboratories among four groups of 275, 268, 317, and 304 participants across China from 2011 through 2012. A total of eight commercial kits were used for C. trachomatis PCR detection in participants.
Nine laboratories reported false-positive results (0.9%). As the series dilution increased, the correct reporting of the data sets decreased; the lowest correct rate was 96.3% in the weakest positive samples (104 copies/mL). Eight laboratories reported false-positive results, and 42 laboratories reported false-negative results in the EQA detection of C. trachomatis. No significant differences were observed in the detection of the constructed C. trachomatis positive samples (97.9%, 98.5%, 100%, 98.5%; P=0.36) and negative samples (100%, 99.0%, 100%, 99.0%; P=0.764) using four commercial kits commonly used in China.
The results of the EQA study indicated that the constructed material provides a noninfectious, stable control material with sufficient volume for PCR detection of C. trachomatis.
Annals of Laboratory Medicine 09/2014; 34(5):360-6. DOI:10.3343/alm.2014.34.5.360 · 1.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
This study aimed at analyzing the prevalence of hepatitis B virus (HBV) DNA among hepatitis B surface antigen (HBsAg)–negative donations by cobas TaqScreen MPX test (Roche Molecular Systems) and discussing the meaning of a reactive minipool (MP) that does not resolve to an individual donation (ID)–reactive result.Study Design and Methods
Nucleic acid amplification testing (NAT) was performed in 12 Chinese blood centers on 826,044 serologic negative donations in MPs of six. MP-reactive pools that were resolved to ID-reactive donations were confirmed by Roche TaqMan viral load assays. Antibody to hepatitis B surface antigen and antibody to hepatitis B core antigen (anti-HBc) results were also analyzed. Cycle threshold (Ct) values of reactive MPs were analyzed in relation to the probability of pool resolution.ResultsA total of 1267 of 137,674 pools were reactive, of which 839 donations were reactive by ID-NAT. The MP6 HBV NAT–yield rate lay between 1 in 1600 and 1 in 1000. At MP Ct values equal or below 37, the probability of pool resolution was approximately 80%. The prevalence of anti-HBc in ID-reactive donations was 81%. The proportion of reactive pools that could not be resolved was 36%. The prevalence of anti-HBc in donations implicated in nonresolved MPs was significantly higher than those in nonreactive MPs (48% vs. 37%, p = 0.016).Conclusion
The anti-HBc data suggest that approximately 10% of nonresolved MPs contain HBV DNA from a low-viral-load occult carrier. We consider ID-NAT resolution testing in duplicate to minimize HBV transmission risk associated with transfusing nonreactive donations implicated in reactive MPs.
[Show abstract][Hide abstract] ABSTRACT: Context: As a new antibody concept, natural bispecific antibodies (nBsAbs) have been detected in long-term passive immunization and some diseases, but their potential immunomodulatory role remains unclear. Hashimoto thyroiditis (HT) appears to fulfill the condition for nBsAb production but has not yet been characterized. Objective: To identify a new nBsAb against thyroid peroxidase (TPO) and thyroglobulin (Tg) in HT patients and to preliminarily explore its immunomodulatory role. Design, Setting, Patients: Serum samples were obtained from 136 HT patients, 92 diseased controls and 99 healthy controls for anti-TPO/Tg nBsAb detection. The relationship between anti-TPO/Tg nBsAb and other clinical parameters was also analyzed. Main Outcome Measures: The anti-TPO/Tg nBsAb was detected using a double-antigen sandwich enzyme-linked immunosorbent assay. Higher nBsAb levels were found to be associated with decreased inflammation in HT patients. Results: The prevalence of anti-TPO/Tg nBsAb in HT was 44.9% (61/136), significantly higher than that of diseased controls (2.2%, 2/92) (p < 0.0001) and healthy controls (0%, 0/99) (p < 0.0001). HT patients who were nBsAb-positive were prone to have significantly lower levels of serum C-reactive protein and tumor necrosis factor alpha compared to the nBsAb-negative individuals (p < 0.05). The serum amyloid A and interferon gamma levels also showed a similar trend in the two groups. The IgG subclass of anti-TPO/Tg nBsAb was IgG4. Further analysis showed a negative correlation between anti-TPO/Tg nBsAb and serum total IgG4 (r = -0.697, p = 0.025) in IgG4 thyroiditis patients. Conclusions: A new type of nBsAb against TPO and Tg in HT patients is identified. Our data also indicate a protective effect of anti-TPO/Tg nBsAb in the pathogenesis of HT and extend prior knowledge about nBsAb in diseases.
[Show abstract][Hide abstract] ABSTRACT: Prostate cancer (PCa) is the most diagnosed cancer in the western male population with high mortality. Recently, alternative approaches based on immunotherapy including mRNA vaccines for PCa have shown therapeutic promise. However, for mRNA vaccine, several disadvantages such as the instability of mRNA, the high cost of gold particles, the limited production scale for mRNA-transfected dendritic cells in vitro, limit their development. Herein, recombinant bacteriophage MS2 virus-like particles (VLPs), which based on the interaction of a 19-nucleotide RNA aptamer and the coat protein of bacteriophage MS2, successfully addressed these questions, in which target mRNA was packaged by MS2 capsid. MS2 VLP-based mRNA vaccines were easily prepared by recombinant protein technology, nontoxic and RNase-resistant. We show the packaged mRNA was translated into protein as early as 12 hr after phagocytosed by macrophages. Moreover, MS2 VLP-based mRNA vaccines induced strong humoral and cellular immune responses, especially antigen-specific cytotoxic T-lymphocyte (CTL) and balanced Th1/Th2 responses without upregulation of CD4(+) regulatory T cells, and protected C57BL/6 mice against PCa completely. As a therapeutic vaccine, MS2 VLP-based mRNA vaccines delayed tumor growth. Our results provide proof of concept on the efficacy and safety of MS2 VLP-based mRNA vaccine, which provides a new delivery approach for mRNA vaccine and implies important clinical value for the prevention and therapy of PCa.
International Journal of Cancer 04/2014; 134(7). DOI:10.1002/ijc.28482 · 5.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An external quality assessment (EQA) program for molecular detection of avian influenza A (H7N9) virus was implemented by National Center for Clinical Laboratory (NCCL) of China during June 2013. Virus-like particles (VLPs) that containing full length RNA sequences of hemagglutinin (HA), neuraminidase (NA), matrix protein (MP) and nucleoprotein (NP) genes from the H7N9 virus (armored RNAs) were constructed. The EQA panel comprising 6 samples with different concentrations of armored RNAs positive for H7N9 viruses and four H7N9-negative samples (including one sample positive only for MP gene of the H7N9 virus) was distributed to 79 laboratories in China that carry out molecular diagnosis of H7N9 viruses. The overall performances of data sets were classified according to the results of both H7 and N9. Consequently, 80 data sets were received (one participant provided two sets of results), which were generated using commercial (n = 60) or in-house (n = 17) real-time RT-PCR (qRT-PCR) kits and commercial assay employed isothermal amplification method (n =3). The results revealed that the majority (82.5%) of data sets correctly identified "H7N9 virus" while 17.5% of the data sets need to improve their diagnostic capability. The "improvable" data sets were derived mostly from false-negative results of N9 at relatively low concentrations. The false-negative rate was 5.6% and the false-positive rate was 0.6%. In addition, we observed varied diagnostic capabilities between different commercially available kits and in-house developed assays, with assay manufactured by BioPerfectus Technologies (Jiangsu, China) performing better than others did. Overall, the majority of laboratories have reliable diagnostic capacity.
[Show abstract][Hide abstract] ABSTRACT: Remarkable progress has been made in the quality assurance of Hepatitis B virus (HBV) DNA nucleic amplification techniques (NAT) during the past decade. And this report presents a 10-y external quality assessment (EQA) program performed by National Center for Clinical Laboratories in China since 2003.
EQA panels were produced using freeze-dried HBV plasma or negative controls and then calibrated against the first International Standard for HBV DNA.
By 2012, total 35,570 qualitative EQA reports and 56,826 quantitative reports have been collected. The overall correct recognition rate in qualitative test increased from 95.15% in 2003 to 97.99% in 2012. The proportion of participants with acceptable quantitative results also rose to 87.99% in 2012 compared with that of 27.53% in 2003. Besides, we observed a satisfactory reproducibility of <5% in all parallel samples. However, some laboratories still had difficulties in exact quantification of some low viral loads, which near to the limits of the dynamic range of the assays.
Taking together, current EQA program showed an encouraging improvement of HBV DNA NAT in China. Distributing more challenging samples and increasing the subtypes are still needed in the future.
Clinica chimica acta; international journal of clinical chemistry 08/2013; 425. DOI:10.1016/j.cca.2013.07.026 · 2.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: In China, various assays for human papillomavirus (HPV) genotyping are currently used for cervical cancer screening. However, a proficiency test system is not available for standardizing and evaluating assay performance. The aim of this study was to evaluate the performance of clinical laboratories for their ability to discriminate 9 HPV types with the proficiency panel. Methods: The panel of 24 samples included cloned genomic DNAs for HPV types 6, 11, 16, 18, 31, 33, 39, 51 and 52 at different concentrations, which were distributed to 76 clinical laboratories. Results reported by participants were compared with the reference results. Results: The samples containing 10(6) IU HPV-16/ml and 10(6) IU HPV-18/ml were (98.7 and 96.0%, respectively) identified correctly most often. For other high-risk HPV types, about 90% of data sets correctly identified samples containing 10(6) GE/ml of HPV-6, HPV-31, HPV-33 and HPV-52, while HPV-51, HPV-11 and HPV-39 in 10(6) GE/ml were correctly identified by only 42.7, 55.6 and 21.3% of laboratories, respectively. Conclusion: Our proficiency test system provided a traceable panel and showed that the differences in performance between laboratories were high, indicating that it is necessary for the laboratories to improve their operation and standardization of HPV genotyping.
[Show abstract][Hide abstract] ABSTRACT: Background
Food allergy has been reported increasingly around the world during the past several decades. Epstein-Barr virus (EBV), a common herpesvirus with high infection rate, is now suspected to be a risk or protective factor in food allergy. The aim of the study was to investigate the possible role of EBV infection in IgE-mediated food allergy.
34 patients with an egg allergy and 34 healthy controls participated in this study. Egg allergy was confirmed by open-food challenge. Serum anti-viral capsid antigen (VCA), anti-Epstein-Barr nuclear antigen 1 (EBNA-1) IgG and egg specific (yolk and white)-IgE levels were evaluated by enzyme linked immunosorbent assay (ELISA). At the same time, EBV DNA as well as viral miRNAs in these samples was quantified by real-time PCR.
The results showed that serum anti EBNA-1 IgG and two viral miRNAs (miR-BART1-5p and miR-BART7) were highly expressed in patients with egg allergy compared with healthy controls (p < 0.05, < 0.001 and < 0.01, respectively). Moreover, the expressions of anti EBNA-1 specific IgG, miR-BART1-5p and miR-BART7 positively correlated with the level of egg-specific IgE (p < 0.05, < 0.01 and < 0.01, respectively). The differences in anti VCA IgG concentration and EBV DNA copy number between the allergy patients and control individuals were not statistically significant.
The high expression of EBV-specific antibody and miRNAs indicated that EBV infection might play a promoting role in IgE-mediated egg food allergy, and viral miRNAs-related immunomodulatory pathway was likely involved in this allergy process.
[Show abstract][Hide abstract] ABSTRACT: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the presence of pathogenic autoantibodies. Recent studies suggest that microRNAs (miRNAs) play an essential role in immunoregulation and may be involved in the pathogenesis of SLE. Therefore, it was of interest to investigate the potential therapeutic application of miRNAs in SLE, a concept that has not been thoroughly investigated thus far. Virus-like particles (VLPs) are a type of recombinant nanoparticle enveloped by certain proteins derived from the outer coat of a virus. Herein, we describe a novel miRNA-delivery approach via bacteriophage MS2 VLPs and investigate the therapeutic effects of miR-146a, a well-studied and SLE-related miRNA, in BXSB lupus-prone mice.
VLPs containing miR-146a, and the control VLPs, were prepared using an Escherichia coli expression system and then administered to lupus-prone mice over a 12-day period. We performed an enzyme-linked immunosorbent assay to evaluate the anti-dsDNA antibody, autoantibody to nuclear antigen (ANA), total IgG and total IgM levels in serum. The expression of miR-146a was analyzed by qRT-PCR. SLE-related cytokines as well as some toll-like receptor signaling pathway molecules were also measured.
Treatment with MS2-miR146a VLP showed profound effects on lupus-prone BXSB mice, including an increased level of mature miR-146a, which led to a significant reduction in the expression of autoantibodies and total IgG. Remarkably, these mice also exhibited reduced levels of proinflammatorycytokines, including IFN-Interferon-α (IFN-α), Interleukin-1β (Il-1β) and Interleukin-6 (Il-6). Moreover, we showed that the toll-like receptor pathway was involved in this regulation.
Restoring the loss of miR-146a was effective in eliminating the production of autoantibodies and ameliorating SLE progression in lupus-prone mice. Thus, the induction of dysregulated miRNAs by an MS2 VLP-based delivery system may lead to novel therapies.
International Journal of Nanomedicine 12/2012; 7:5957-67. DOI:10.2147/IJN.S37990 · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To construct and express ribonuclease-resistant virus-like particles containing rotavirus NSP3 gene by changing the affinity of MS2 bacteriophage coat protein pac site and to discuss the stability.
In the study, 1 049 bp rotavirus NSP3 gene fragments were amplified by PCR using the primers containing PvuI and KpnI restriction enzyme sites and the uridine at position -5 in the pac site was replaced with cytosine to increase the affinity. The gel-purified PCR-amplified DNA fragments and pACYC-MS2 vector were digested with PvuI and KpnI and then ligated to generate recombinant plasmid pACYC-MS2-NSP3. The expression vector was transformed into competent Escherichia coli strain TOP 10, and was verified by PCR and sequencing. The positive bacteria were transformed into competent E.coli strain BL21(DE3). Then the cells were lysed by ultrasonic disruption, virus like particles (VLPs) were harvested after purification and their stability was discussed.
The expression plasmid containing mutant pac site (uridine at position -5 in the pac site replaced with cytosine) was constructed successfully and the ribonuclease-resistant virus-like particles containing rotavirus NSP3 gene were expressed successfully. The VLPs were resistant to ribonuclease and deoxyribonuclease, and were stable at -20 °C, 4 °C and room temperature(25 °C), respectively.
The methods used to increase the affinity of pac site could successfully construct and express the VLPs. The VLPs containing rotavirus NSP3 gene are stable and could be used as surrogates for positive controls and standards in rotavirus real time fluorescent quantitative reverse transcription-PCR kits.
Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences 10/2012; 44(5):737-41.
[Show abstract][Hide abstract] ABSTRACT: Abstract During the past few years there has been great interest in the development of circulating microRNAs (miRNAs) as stable blood-based biomarkers for cancer detection. Deregulation of miRNAs in blood samples has shown considerable clinical utilities in cancers. Due to poorly characterized preanalytical and analytical variables and the lack of a standardized measurement protocol, the application of these miRNA fingerprints is hindered by conflicting results. In this review, we outline our current understanding of preanalytically and analytically confounding factors. We believe that great consideration should be taken in the development of circulating miRNA as tumor biomarkers.
Clinical Chemistry and Laboratory Medicine 09/2012; 50(12):1-6. DOI:10.1515/cclm-2012-0360 · 2.96 Impact Factor