Objective To analyze the genetic subtypes of human immunodeficiency virus (HIV) and the prevalence of pretreatment drug resistance in the newly reported HIV-infected men in Guangxi. Methods The stratified random sampling method was employed to select the newly reported HIV-infected men aged≥50 years old in 14 cities of Guangxi from January to June in 2020.The pol gene of HIV-1 was amplified by nested reverse transcription polymerase chain reaction and then sequenced.The mutation sites associated with drug resistance and the degree of drug resistance were then analyzed. Results A total of 615 HIV-infected men were included in the study.The genetic subtypes of CRF01_AE,CRF07_BC,and CRF08_BC accounted for 57.4% (353/615),17.1% (105/615),and 22.4% (138/615),respectively.The mutations associated with the resistance to nucleoside reverse transcriptase inhibitors (NRTI),non-nucleoside reverse transcriptase inhibitors (NNRTI),and protease inhibitors occurred in 8 (1.3%),18 (2.9%),and 0 patients,respectively.M184V (0.7%) and K103N (1.8%) were the mutations with the highest occurrence rates for the resistance to NRTIs and NNRTIs,respectively.Twenty-two (3.6%) patients were resistant to at least one type of inhibitors.Specifically,4 (0.7%),14 (2.3%),4 (0.7%),and 0 patients were resistant to NRTIs,NNRTIs,both NRTIs and NNRTIs,and protease inhibitors,respectively.The pretreatment resistance to NNRTIs had much higher frequency than that to NRTIs (2.9% vs.1.3%;χ²=3.929,P=0.047).The prevalence of pretreatment resistance to lamivudine,zidovudine,tenofovir,abacavir,rilpivirine,efavirenz,nevirapine,and lopinavir/ritonavir was 0.8%, 0.3%, 0.7%, 1.0%, 1.3%, 2.8%, 2.9%, and 0, respectively. Conclusions CRF01_AE,CRF07_BC,and CRF08_BC are the three major strains of HIV-infected men≥50 years old newly reported in Guangxi,2020,and the pretreatment drug resistance demonstrates low prevalence.
Male ; Humans ; Middle Aged ; Reverse Transcriptase Inhibitors/therapeutic use* ; HIV Infections/drug therapy* ; Drug Resistance, Viral/genetics* ; China/epidemiology* ; Mutation ; HIV-1/genetics* ; Protease Inhibitors/therapeutic use* ; Genotype

OBJECTIVE: To establish a rapid detection and genotyping method for SARS-CoV-2 Omicron BA.4/5 variants using CRISPPR-Cas12a gene editing technology. METHODS: We combined reverse transcription-polymerase chain reaction (RT-PCR) and CRISPR gene editing technology and designed a specific CRISPPR RNA (crRNA) with suboptimal protospacer adjacent motifs (PAM) for rapid detection and genotyping of SARS- CoV-2 Omicron BA.4/5 variants. The performance of this RT- PCR/ CRISPPR-Cas12a assay was evaluated using 43 clinical samples of patients infected by wild-type SARS-CoV-2 and the Alpha, Beta, Delta, Omicron BA. 1 and BA. 4/5 variants and 20 SARS- CoV- 2-negative clinical samples infected with 11 respiratory pathogens. With Sanger sequencing method as the gold standard, the specificity, sensitivity, concordance (Kappa) and area under the ROC curve (AUC) of RT-PCR/CRISPPR-Cas12a assay were calculated. RESULTS: This assay was capable of rapid and specific detection of SARS- CoV-2 Omicron BA.4/5 variant within 30 min with the lowest detection limit of 10 copies/μL, and no cross-reaction was observed in SARS-CoV-2-negative clinical samples infected with 11 common respiratory pathogens. The two Omicron BA.4/5 specific crRNAs (crRNA-1 and crRNA-2) allowed the assay to accurately distinguish Omicron BA.4/5 from BA.1 sublineage and other major SARS-CoV-2 variants of concern. For detection of SARS-CoV-2 Omicron BA.4/5 variants, the sensitivity of the established assay using crRNA-1 and crRNA-2 was 97.83% and 100% with specificity of 100% and AUC of 0.998 and 1.000, respectively, and their concordance rate with Sanger sequencing method was 92.83% and 96.41%, respectively. CONCLUSION By combining RT-PCR and CRISPPR-Cas12a gene editing technology, we successfully developed a new method for rapid detection and identification of SARS-CoV-2 Omicron BA.4/5 variants with a high sensitivity, specificity and reproducibility, which allows rapid detection and genotyping of SARS- CoV-2 variants and monitoring of the emerging variants and their dissemination.
Humans ; COVID-19 ; CRISPR-Cas Systems ; Genotype ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; SARS-CoV-2/genetics* ; RNA ; COVID-19 Testing

OBJECTIVE: This study aimed to determine the HIV-1 subtype distribution and HIV drug resistance (HIVDR) in patients with ART failure from 2014 to 2020 in Hainan, China. METHODS: A 7-year cross-sectional study was conducted among HIV/AIDS patients with ART failure in Hainan. We used online subtyping tools and the maximum likelihood phylogenetic tree to confirm the HIV subtypes with pol sequences. Drug resistance mutations (DRMs) were analyzed using the Stanford University HIV Drug Resistance Database. RESULTS: A total of 307 HIV-infected patients with ART failure were included, and 241 available pol sequences were obtained. Among 241 patients, CRF01_AE accounted for 68.88%, followed by CRF07_BC (17.00%) and eight other subtypes (14.12%). The overall prevalence of HIVDR was 61.41%, and the HIVDR against non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleotide reverse transcriptase inhibitors (NRTIs), and protease inhibitors (PIs) were 59.75%, 45.64%, and 2.49%, respectively. Unemployed patients, hypoimmunity or opportunistic infections in individuals, and samples from 2017 to 2020 increased the odd ratios of HIVDR. Also, HIVDR was less likely to affect female patients. The common DRMs to NNRTIs were K103N (21.99%) and Y181C (20.33%), and M184V (28.21%) and K65R (19.09%) were the main DRMs against NRTIs. CONCLUSION The present study highlights the HIV-1 subtype diversity in Hainan and the importance of HIVDR surveillance over a long period.
Humans ; Reverse Transcriptase Inhibitors/therapeutic use* ; HIV-1/genetics* ; Cross-Sectional Studies ; Phylogeny ; Anti-HIV Agents/therapeutic use* ; Drug Resistance, Viral/genetics* ; HIV Infections/epidemiology* ; Mutation ; China/epidemiology* ; Prevalence ; Genotype

OBJECTIVE: To establish a new digital polymerase chain reaction (dPCR) system for the detection of BCR-ABL fusion gene in patients with chronic myeloid leukemia (CML), and explore its analytical performance and clinical applicability in the detection of BCR-ABL^p190/210/230. METHODS: A new dPCR system for detecting BCR-ABL^p190/210/230 was successfully developed, and its sensitivity difference with qPCR and improvement of drug side effects in patients with CML during drug reduction or withdrawal were compared. RESULTS: Among 176 samples, qPCR and dPCR showed high consistency in the sensitivity of detecting BCR-ABL (82.39%), and the positive rate of dPCR was about 5 times higher that of qPCR (20.45% vs 3.98%). During follow-up, blood routine (25% vs 10%), kidney/liver/stomach (25% vs 20%) and cardiac function (10% vs 0) were significantly improved after drug reduction or withdrawal in patients with initial dPCR negative compared with before drug reduction or withdrawal. CONCLUSIONS This new dPCR detection system can be applied to the detection of BCR-ABL^p190/210/230. It has better consistency and higher positive detection rate than qPCR. Drug withdrawal or dose reduction guided by dPCR has a certain effect on improving drug side effects.
Humans ; Fusion Proteins, bcr-abl/genetics* ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis* ; Polymerase Chain Reaction ; Drug-Related Side Effects and Adverse Reactions ; Reverse Transcriptase Polymerase Chain Reaction

Objective: To evaluate the application of real-time RT-PCR and semi-nested RT-PCR in the detection of norovirus in oysters and analyzing the genetic characteristics of the isolates. Methods: Real-time fluorescent RT-PCR and semi-nested RT-PCR were used to detect norovirus GⅠ/GⅡ in fresh oysters collected from the markets in Beijing from November 2014 to October 2015. The detection rate of the parallel test was also analyzed. In addition, the reliability of semi-nested RT-PCR was evaluated by agreement rate and consistency test (Kappa value). The positive products of norovirus GⅠ/GⅡ capsid protein region gene by semi-nested RT-PCR were sequenced. Software BioEdit 7.0.9.0 was used for sequence alignment, and software Mega 6.0 was used to construct the evolutionary tree. Results: In 72 samples, the detection rate of norovirus was 31.94% (23/72) by real-time RT-PCR, 38.89% (28/72) by semi-nested RT-PCR and 48.61% (35/72) by parallel test. The coincidence rate of the two methods was 73.61%, a moderate degree (Kappa value =0.43). A total of 13 norovirus strains were successfully sequenced, and 11 strains (7 GⅡ.17 strains, 2 GⅡ. 4 Sydney_ 2012 strains, 1 GⅡ. 1 strain and 1 GⅡ. 21 strain) were obtained from norovirus positive samples by two RT-PCR methods, two strains (1 GⅡ. 17 strain and 1 GⅡ. 3 strain) were obtained from real-time RT-PCR negative samples which were positive for norovirus by semi-nested RT-PCR. The similarity between these strains and reference strains from diarrhea patients, environmental sewage, and shellfish products were 84.4% - 100.0%. Conclusions: The parallel test of norovirus in oysters by two RT-PCR methods can improve the detection rate and detect more genotypes. Norovirus strains in oysters were highly homologous with reference strains from diarrheal patients, environmental sewage, and shellfish products. Therefore, surveillance, prevention and control for norovirus should be carried out in people who have frequent contacts with oysters and related environments.
Animals ; Beijing ; Humans ; Norovirus/genetics* ; Ostreidae ; RNA, Viral/genetics* ; Real-Time Polymerase Chain Reaction ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction

Objective: To investigate the distribution of HIV-1 genetic subtypes and pretreatment drug resistance (PDR) among men who have sex with men (MSM) from 19 cities of 6 provinces in China. Methods: From April to November 2019, 574 plasma samples of ART-naive HIV-1 infected MSM were collected from 19 cities in Hebei, Shandong, Jiangsu, Zhejiang, Fujian, and Guangdong provinces, total ribonucleic acid (RNA) was extracted and amplified the HIV-1 pol gene region by nested polymerase chain reaction (PCR) after reverse transcription. Then sequences were used to construct a phylogenetic tree to determine genetic subtypes and submitted to the Stanford drug resistance database for drug resistance analysis. Results: A total of 479 samples were successfully amplified by PCR. The HIV-1 genetic subtypes included CRF01_AE, CRF07_BC, B, CRF55_01B, CRF59_01B, CRF65_cpx, CRF103_01B, CRF67_01B, CRF68_01B and unrecognized subtype, which accounted for 43.4%, 36.3%, 6.3%, 5.9%, 0.8%, 0.8%, 0.4%, 0.4%, 0.2% and 5.5%, respectively. The distribution of genetic subtypes among provinces is statistically different (χ²=44.141, P<0.001). The overall PDR rate was 4.6% (22/479), the drug resistance rate of non-nucleoside reverse transcriptase inhibitors, nucleoside reverse transcriptase inhibitors, and protease inhibitors were 3.5% (17/479), 0.8% (4/479) and 0.2% (1/479), respectively. The PDR rate of recent infections was significantly higher than that of long-term infections (χ²=4.634, P=0.031). Conclusions: The HIV-1 genetic subtypes among MSM infected with HIV-1 from 19 cities of 6 provinces in China are diverse, and the distribution of subtypes is different among provinces. The overall PDR rate is low, while the PDR rate of recent infections was significantly higher than that of long-term infections, suggesting the surveillance of PDR in recent infections should be strengthened.
China/epidemiology* ; Cities ; Drug Resistance ; Drug Resistance, Viral/genetics* ; Female ; Genotype ; HIV Infections/epidemiology* ; HIV Seropositivity/drug therapy* ; HIV-1/genetics* ; Homosexuality, Male ; Humans ; Male ; Phylogeny ; Reverse Transcriptase Inhibitors/therapeutic use* ; Sexual and Gender Minorities

BACKGROUND: Arsenic is a developmental neurotoxicant. It means that its neurotoxic effect could occur in offspring by maternal arsenic exposure. Our previous study showed that developmental arsenic exposure impaired social behavior and serotonergic system in C3H adult male mice. These effects might affect the next generation with no direct exposure to arsenic. This study aimed to detect the social behavior and related gene expression changes in F2 male mice born to gestationally arsenite-exposed F1 mice. METHODS: Pregnant C3H/HeN mice (F0) were given free access to tap water (control mice) or tap water containing 85 ppm sodium arsenite from days 8 to 18 of gestation. Arsenite was not given to F1 or F2 mice. The F2 mice were generated by mating among control F1 males and females, and arsenite-F1 males and females at the age of 10 weeks. At 41 weeks and 74 weeks of age respectively, F2 males were used for the assessment of social behavior by a three-chamber social behavior apparatus. Histological features of the prefrontal cortex were studied by ordinary light microscope. Social behavior-related gene expressions were determined in the prefrontal cortex by real time RT-PCR method. RESULTS: The arsenite-F2 male mice showed significantly poor sociability and social novelty preference in both 41-week-old group and 74-week-old group. There was no significant histological difference between the control mice and the arsenite-F2 mice. Regarding gene expression, serotonin receptor 5B (5-HT 5B) mRNA expression was significantly decreased (p < 0.05) in the arsenite-F2 male mice compared to the control F2 male mice in both groups. Brain-derived neurotrophic factor (BDNF) and dopamine receptor D1a (Drd1a) gene expressions were significantly decreased (p < 0.05) only in the arsenite-F2 male mice of the 74-week-old group. Heme oxygenase-1 (HO-1) gene expression was significantly increased (p < 0.001) in the arsenite-F2 male mice of both groups, but plasma 8-hydroxy-2'-deoxyguanosine (8-OHdG) and cyclooxygenase-2 (COX-2) gene expression were not significantly different. Interleukin-1β (IL-1β) mRNA expression was significantly increased only in 41-week-old arsenite-F2 mice. CONCLUSIONS These findings suggest that maternal arsenic exposure affects social behavior in F2 male mice via serotonergic system in the prefrontal cortex. In this study, COX-2 were not increased although oxidative stress marker (HO-1) was increased significantly in arsnite-F2 male mice.
Animals ; Arsenic/toxicity* ; Arsenites/toxicity* ; Behavior, Animal/drug effects* ; Environmental Pollutants/toxicity* ; Female ; Gene Expression/drug effects* ; Genetic Markers ; Male ; Maternal Exposure/adverse effects* ; Mice ; Mice, Inbred C3H ; Oxidative Stress/genetics* ; Prefrontal Cortex/drug effects* ; Pregnancy ; Prenatal Exposure Delayed Effects/psychology* ; Reverse Transcriptase Polymerase Chain Reaction ; Serotonin/metabolism* ; Social Behavior ; Sodium Compounds/toxicity*

BACKGROUND: With the ongoing worldwide coronavirus disease 2019 (COVID-19) pandemic, an increasing number of viral variants are being identified, which poses a challenge for nucleic acid-based diagnostic tests. Rapid tests, such as real-time reverse transcription-polymerase chain reaction (rRT-PCR), play an important role in monitoring COVID-19 infection and controlling its spread. However, the changes in the genotypes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants may result in decreased sensitivity of the rRT-PCR assay and it is necessary to monitor the mutations in primers and probes of SARS-CoV-2 detection over time. METHODS: We developed two rRT-PCR assays to detect the RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) genes of SARS-CoV-2. We evaluated these assays together with our previously published assays targeting the ORF1ab and N genes for the detection and confirmation of SARS-CoV-2 and its variants of concern (VOCs). In addition, we also developed two rRT-PCR assays (S484K and S501Y) targeting the spike gene, which when combined with the open reading frames (ORF)1ab assay, respectively, to form duplex rRT-PCR assays, were able to detect SARS-CoV-2 VOCs (lineages B.1.351 and B.1.1.7). RESULTS: Using a SARS-CoV-2 stock with predetermined genomic copies as a standard, the detection limit of both assays targeting RdRp and N was five copies/reaction. Furthermore, no cross-reactions with six others human CoVs (229E, OC43, NL63, HKU1, severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus) were observed using these assays. In addition, the S484K and S501Y assays were combined with the ORF1ab assay, respectively. CONCLUSIONS Four rRT-PCR assays (RdRp, N, S484K, and S501Y) were used to detect SARS-CoV-2 variants, and these assays were shown to be effective in screening for multiple virus strains.
COVID-19 ; Humans ; RNA, Viral/genetics* ; Real-Time Polymerase Chain Reaction ; Reverse Transcription ; SARS-CoV-2 ; Sensitivity and Specificity

Screening suitable reference genes is the premise of quantitative Real-time PCR(qRT-PCR)for gene expression analysis. To provide stable reference genes for expression analysis of genes in Aconitum vilmorinianum, this study selected 19 candidate re-ference genes(ACT1, ACT2, ACT3, aTUB1, aTUB2, bTUB, 18S rRNA, UBQ, eIF2, eIF3, eIF4, eIF5, CYP, GAPDH1, GAPDH2, PP2A1, PP2A2, ACP, and EF1α) based on the transcriptome data of A. vilmorinianum. qRT-PCR was conducted to profile the expression of these genes in the root, stem, leaf, and flower of A. vilmorinianum. The Ct values showed that 18S rRNA with high expression level and GAPDH2 with large expression difference among organs were not suitable as the reference genes. NormFinder and geNorm showed similar results of the expression stability of the other candidate reference genes and demonstrated PP2A1, EF1α, and CYP as the highly stable ones. However, BestKeeper suggested EF1α, ACT3, and PP2A1 as the top stable genes. In view of the different results from different softwares, the geometric mean method was employed to analyze the expression stability of the candidate re-ference genes, the results of which indicated that PP2A1, EF1α, and ACT3 were the most stable. Based on the comprehensive analysis results of geNorm, NormFinder, BestKeeper, and geometric mean method, PP2A1 and EF1α presented the most stable expression in different organs of A. vilmorinianum. PP2A1 and EF1α were the superior reference genes for gene expression profiling in different organs of A. vilmorinianum.
Aconitum ; Gene Expression Profiling ; Genes, Plant/genetics* ; Real-Time Polymerase Chain Reaction ; Reference Standards ; Reverse Transcriptase Polymerase Chain Reaction

Freshly collected seeds of Amomum tsaoko demonstrate obvious dormancy. Therefore, the selection of stable reference genes during seed dormancy release is very important for the subsequent functional research of related genes. In this study, ten commonly used reference genes(GAPDH, 40S, actin, tubulin, EIF4A-9, EIF2α, UBC, UBCE2, 60S, and UBQ) were selected as candidates for quantitative Real-time polymerase chain reaction(qRT-PCR) of the embryo samples of A. tsaoko at different dormancy release stages. Three kinds of software(BestKeeper, geNorm, and Normfinder) and the Delta CT method were used to evaluate the expression stability of the candidate reference genes, and the RefFinder online tool was employed to integrate the results and generate a comprehensive ranking. The results showed that the expression levels of the ten candidate reference genes differed greatly in different embryo samples. GAPDH and UBC had high expression levels, as manifested by the small Ct values. GeNorm identified 40S and UBCE2 as the most stable genes. NormFinder ranked EIF2α as the most stable gene and UBC as the least stable gene. UBCE2 was found to be the most stable gene and actin the least stable one by BestKeeper. Delta CT analysis suggested that the expression of 40S was most stable. UBCE2 was recommended as the most stably expressed gene by RefFinder. Thus, UBCE2 is the ideal reference gene for qRT-PCR analysis of A. tsaoko seeds at different dormancy release stages. The results may lay a foundation for analyzing the expression of related genes during seed dormancy release of A. tsaoko.
Amomum ; Gene Expression Profiling ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Seeds/genetics*