Challenges and considerations on quality control and evaluation of pathogen metagenomic next-generation sequencing / 生物工程学报

Metagenomic next-generation sequencing (mNGS) could be used for pathogen detection from nearly all types of clinical samples. Especially, the unique diagnostic capability of pathogen mNGS detecting unknown causative agent of infectious diseases makes this method become an importation complement and irreplaceable component for conventional routine laboratory test. However, the complexity of the testing process, the rapid product update, and the insufficiency in quality control and evaluation methods that all make clinical transformation, industry development, and regulation of this technology full of challenge and uncertainty. This review briefly introduces the technical advantages and challenges, and describes the general workflow and quality control steps in details. Finally, it focuses on current considerations regarding quality evaluation methods and standards for pathogen mNGS.

Evaluation of HIV diagnosis kits used for blood screening with NIFDC-established HIV-1 seroconver-sion panels / 中华微生物学和免疫学杂志

Objective To establish HIV-1 seroconversion panels with the samples collected by National Institutes for Food and Drug Control ( NIFDC) and to evaluate the window periods of HIV enzyme immunoassay ( EIA) diagnostic kits used for blood screening with them. Methods Serum specimens were collected from different plasma donation stations in China. All suspected HIV infection specimens were screened for HIV by using the nucleic acid amplification testing (NAT), Western blot confirmatory assay and P24 quantitative detection assay. The HIV env gene sequences were amplified by RT-PCR for further confirmation of HIV infection. The PCR products were sequenced and genotyped. The confirmed seroconver-sion panels were used to evaluate the early detection capabilities of the 4th and 3rd generation HIV EIA diag-nostic kits used in blood screening in china. Results A total of 8 sets of HIV seroconversion panels com-prised of 36 samples were confirmed in this study, including 6 sets of AE subtype, 1 set of B subtype and 1 set of unknown genotype. Those seroconversion panels were tested with HIV diagnostic kits produced by 19 different manufacturers. For the early detection of HIV infection, the 4th generation HIV diagnostic kits with a score of 9. 4 points were better than the 3rd generation HIV diagnostic kits whose score was 3. 6 points (P<0. 01, t=8. 547). Some of the domestic 4th generation HIV diagnostic kits were similar to the imported kits in the early detection of HIV infection. In terms of the diagnosis of HIV infection, the HIV-1 NAT was at least 2 weeks earlier than the HIV EIA diagnostic kits. The sensitivity of confirmatory assay was lower than that of the diagnostic kits. Four out of five 4th generation HIV diagnostic kits showed declined signal to cut off ( S/CO ) ratio , indicating the probability of false detection during the second window period . Conclusion Eight sets of NIFDC HIV-1 seroconversion panels were established in this study. With those panels we found that there were differences in the window period between different EIA diagnostic kits used for HIV blood screening.

Establishment and optimization of a high throughput phenotypic test for the detection of drug resist-ance in human immunodeficiency virus(HIV)strains / 中华微生物学和免疫学杂志

Objective To establish a high throughput phenotypic test for the detection of drug re-sistance in human immunodeficiency virus(HIV)strains. Methods The gene encoding luciferase was in-activated through restriction enzyme digestion and ligation. LacZ gene was used to replace the genes encoding original protease and reverse transcriptase. pol genes were amplified from pSG3△env plasmid and cloned in-to a new backbone plasmid through infusion. The factors that might affect the results of the test were opti-mized. Results The parental backbone plasmid pNL4-3. Lac was constructed,of which the gene encoding luciferase was inactivated and bearing the LacZ gene instead of genes encoding protease and reverse tran-scriptase. Several influential factors including cell numbers(10 000 / well),virus inoculation(200 TCID50 /well)and the concentration of DEAE-dextran(15 μg/ ml)were optimized. The reproducibility of this test was confirmed by testing 12 anti-HIV drugs against 2 pseudovirus strains 8 times,presenting the coefficient of variations(CVs)from 4. 32% to 28. 46% . Six types of pseudovirus were constructed and tested against the 12 anti-HIV drugs,the results of which were compared with those by using the pSG3△env-based pseud-ovirus test. The results of the two tests presented good consistency. Conclusion The high throughput phe-notypic test based on pNL4-3. Lac plasmid,combining the advantages of pSG3△env and pNL4-3 systems, could be used to analyze the drug resistance patterns of HIV-1 infectors and screen new drugs for antiretrovi-ral therapy in a rapid and effective way.

Evaluation of four domestic and three imported fourth-generation HIV diagnostic reagents / 中华微生物学和免疫学杂志

Objective To evaluate the quality of four domestic and three imported fourth-generation HIV diagnostic reagents.Methods The specificity and sensitivity of these assays were analyzed when testing HIV negative samples and HIV-1 RNA positive samples.The relative seroconversion sensitivity index was analyzed when testing BBI seroconversion panels.Results The sensitivity of seven 4th-generation assays were 100％ (95％ CI:99.86％-100％ ),and one sample at the window period of HIV-1 infection were detected as positive.Of the seven assays,one imported assay exhibited the relative largeδ + value (1.0892),and the small δ+ value were found on the remaining six assays (0.0836-0.3003 ).For the samples negative for HIV antibody,varying degrees of false positives were observed on the seven assays ( specificity:97.80％ -99.60％,δ- value:-1.3803 to -0.4778).When testing the BBI seroconversion panels,the relative seroconversion sensitivity index of domestic assays were -0.500-0,however,which of imported assays were -0.600 and -0.700.Conclusion The seven reagents exhibited high sensitivity and specificity.The 4th generation HIV assays can be used as blood screening reagents to find the samples at window period of HIV-1 infection,thus indicating the certain meaning in reducing the transmission risk of HIV-1 for fourth-generation HIV diagnostic reagents.However,the better efficiency to detect HIV-1 early infection was observed on the imported assays than on the domestic assays.

Primary researches on human genetically engineered antibodies to HIV-1 / 中华微生物学和免疫学杂志

Objective To pan and characterize anti-HIV-1 Fab by the phage antibody library technology. Methods Total RNA were extracted from lymphocytes which were isolated from peripheral blood collected from asymptomatic HIV-1 infected donors with high titer antibody against HIV-1. The genes of heavy chains Fd fragment and light chains of antibody were amplified by RT-PCR. The phagmids pComb3X cloned Fd and light chain genes were transformed into E. coli XL1-Blue by electroporation to construct phage Fab library. By three runs of "absorption-elution-neutralization-enrichment", the clones were induced by IPTG and characterized by ELISA. The positive clones were sequenced and analyzed the sequences. Subsequently, Fab antibodies of these positive clones were induced to expressed and purified, then the recombinant virus neutralization assay was performed. Results A phage Fab library was constructed with 8×106 members, and 11 positive clones were obtained by detecting IPTG-induced-expressing Fabs with ELISA. By analysis of the sequences, 10 light chain genes and 8 Fd genes were ensured to be obtained. Compared with the genes of anti-HIV-1 antibodies in HIV sequence database, the gene sequences we obtained were highly homologous to some patent genes of anti-HIV-1 gp120 antibodies in HIV sequence database( light chains with 60%-90% identity, Fd with 71%-85% identity); The CDRs of these positive clones were determined by comparing the positive clone genes with antibodies' genes in V base database, furthermore, CDRH3 of these positive clones has the length of 12-22 aa. Strand shift had little effect to improving affinity of our Fab clones. Fab antibodies were induced to express at the concentration of ＞ 10 mg/L. Three Fab antibodies neutralize HIV-1 virus to some extent. Conclusion The studies will provide the basis on further study on the anti-HIV-1 Fabs obtained successfully.

Comparative evaluation of eight third generation and four fourth generation anti-HIV diagnostic reagents / 中华微生物学和免疫学杂志

Objective To evaluate the differences between the third and the fourth generations of anti-HIV assays,and different kits within the same generation.Methods A total of 989 HIV-negative samples,185 samples positive for HIV-1 RNA.1st-generation international references of HIV antibodies and samples from 9 sets of BBI seroconversion panels were detected by 8 kits of the third generation and 4 kits of the fourth.Results The fourth generation kits can detect HIV infection earlier than the third generation kits.However,the detected days of HIV infection with different kits of the fourth generation were different whilst no significant difierences were found with difierent kits of the third generation.Furthermore,the capacity of detecting samples with different genotypes for different reagents was different,especially the capacity of domestic reagents on detecting HIV-1 O group and HIV-2 samples was relatively weak.Conclusion These data provided information to improve the quality of anti-HIV diagnostic reagents further.

EFFECT OF LYCOPENE ON LIFESPAN OF DROSOPHILA AND ITS UNDERLYING MOLECULAR MECHANISM / 营养学报

Objective To explore the effects of lycopene on lifespan in drosophila and the underlying mechanism. Method Drosophila aged 30 d were reared in medium containing 0,2.5,7.5,22.5 ?g/g lycopene till they all die. The average lifespan and the average maximum lifespan were determined. Male drosophila aged 30 d were divided into control group and experiment group and cultured in medium supplemented with 0,22.5 ?g/g lycopene. The gene expression was determined 10,20 d later. Results The average maximum lifespan of male drosophila was increased with 2.5,7.5 ?g/g lycopene supp-lementation(P