A novel multiplex real-time PCR assay for the detection of cytomegalovirus, Epstein-Barr virus, herpes simplex virus 1/2 and strategies for application to blood screening.

A multiplex real-time PCR has been developed to simultaneously detect transfusion-transmissible pathogens cytomegalovirus, Epstein-Barr virus and herpes simplex virus, as well as to provide sample quality testing, for the conserved regions of the cytomegalovirus UL123 gene, the Epstein-Barr virus BKRF1 gene, and the herpes simplex virus 1/2 UL30 gene, tested on 500 blood donors and 320 transfusion recipients. The laboratory sensitivities for all 3 pathogens were 100 copies/µL. Compared to the commercial real-time PCR reference kit, the multiplex real-time PCR assay for the detection of CMV, EBV and HSV presented 100% consistency. In 820 whole blood samples, the multiplex real-time PCR assay identified 34 (4.15%) positive for CMV DNA, 15 (1.83%) positive for EBV DNA, and 6 (0.73%) positive for HSV DNA. For blood transfusions in high-risk groups, whole blood herpes virus test should be included in the spectrum of pathogen testing for blood donors and recipients.

Development and clinical validation of a one-step pentaplex real-time reverse transcription PCR assay for detection of hepatitis virus B, C, E, Treponema pallidum, and a human housekeeping gene.

BACKGROUND: With the safety of blood transfusion being a major public health concern, the development of a rapid, sensitive, specific, and cost-effective multiplex PCR assay for simultaneous detection of hepatitis B virus(HBV), hepatitis C virus (HCV), hepatitis E virus (HEV), and Treponema pallidum(T. pallidum) in blood is crucial. METHODS: Five primer pairs and probes were designed towards conserved regions of target genes and used to establish a one-step pentaplex real-time reverse transcription PCR(qRT-PCR) assay for simultaneous detection of HBV, HCV, HEV, T. pallidum, and RNase P(housekeeping gene), providing sample quality check. The clinical performance of the assay was further determined with 2400 blood samples from blood donors and patients in Zhejiang province, and compared the results with commercial singleplex qPCR and serological assays. RESULTS: The 95% limit of detection(LOD) of HBV, HCV, HEV, and T. pallidum were 7.11 copies/µL, 7.65 copies/µL, 8.45 copies/µL, and 9.06 copies/µL, respectively. Moreover, the assay has good specificity and precision. Compared to the singleplex qPCR assay, the novel assay for detecting HBV, HCV, HEV, and T. pallidum presented 100% clinical sensitivity, specificity, and consistency. Several discrepant results between serological and pentaplex qRT-PCR assays were found. Of 2400 blood samples, there were 2(0.08%) HBsAg positive samples, 3(0.13%) anti-HCV positive samples, 29(1.21%) IgM anti-HEV positive samples and 6(0.25%) anti-T. pallidum positive samples proven negative in nucleic acid detection. 1(0.04%) HBV DNA positive sample and 1(0.04%) HEV RNA positive sample were detected negative by serological testing. CONCLUSIONS: The developed pentaplex qRT-PCR is the first assay on simultaneous, sensitive, specific, and reproducible detection of HBV, HCV, HEV, T. pallidum, and RNase P in a single tube. It could detect pathogens in blood during the window period of infection and is a good tool for effectively screening blood donors and early clinical diagnosis.

Etiological analysis of virus, mycoplasma pneumoniae and chlamydia pneumoniae in hospitalized children with acute respiratory infections in Huzhou.

BACKGROUND: Acute respiratory infections are a common disease in children with high mortality and morbidity. Multiple pathogens can cause acute respiratory infections. A 2-year survey of hospitalized children was conducted to understand the epidemic situation, seasonal spread of pathogens and the improvement of clinical diagnosis, treatment and prevention of disease in Huzhou, China. METHODS: From September 2017 to August 2019, 3121 nasopharyngeal swabs from hospitalized children with acute respiratory infections were collected, and real-time PCR was used to detect various pathogens. Then, pathogen profiles, frequency and seasonality were analyzed. RESULTS: Of the 3121 specimens, 14.45% (451/3121) were positive for at least one pathogen. Of the single-pathogen infections, RSV (45.61%, 182/399) was the most frequent pathogen, followed by PIVs (14.79%, 59/399), ADV (14.54%, 58/399), MP (10.78%, 43/399), and IAV (5.26%, 21/399). Of the 52 coinfections, RSV + PIVs viruses were predominantly identified and accounted for 40.38% (21/52) of cases. RSV was the most frequent pathogen in all four groups. The highest positive rate of the pathogens occurred in the winter (21.26%), followed by autumn (14.98%), the summer (14.11%) and the spring (12.25%). CONCLUSION: Viruses are the main pathogens in hospitalized children with acute respiratory infections in Huzhou city, Zhejiang Province, China. Among the pathogens, RSV had the highest detection rate, and MP is also a common pathogen among children with acute respiratory infections. This study provided a better understanding of the distribution of pathogens in children of different ages and seasons, which is conducive to the development of more reasonable treatment strategies and prevention and control measures.

Anticarcinogenic effect of Umbelliferone in human prostate carcinoma: An in vitro study.

PURPOSE: To explore the chemoprotective effect of umbelliferone (UF) on prostate cancer cell lines, i.e. primary stage (LnCap) and last stage (PC3) prostate cancer together with the effect on the induction of apoptosis and alteration on cell cycle arrest. METHODS: Various concentrations of UF were evaluated against the different prostate cancer cell lines. Lipopolysaccharide (LPS) induced cytokines related factor profiling, proinflammatory cytokines, and inflammatory mediators were studied using Western blot analysis. RESULTS: UF showed significant apoptotic effect. Moreover, treatment with UF did not show apoptosis or cell cycle arrest on the non-cancerous cells including BHP-1, suggesting a selective tumor cell specific effect. UF treatment also enhanced the expression of Bax in PC3 cells, but had no significant effect on the activation of nuclear factor κB (NF-κB). Thus, the apoptosis induction was independent of NF-κB activation. CONCLUSION: The results of the present investigation confirmed the chemoprotective effect of UF in early-stage (Ln- Cap) and late-stage (PC3) prostate cancer cells.

IRF-3 gene polymorphisms are associated with the susceptibility to and the survival in chronic lymphocytic leukemia and could also serve as an auxiliary index.

The objective of this article was to investigate the relationship between IRF-3 gene polymorphisms and the susceptibility and prognosis of CLL. Between January 2011 and August 2012, 108 CLL patients and 112 healthy were enrolled in the study. DHPLC and Shesis software were applied in our study. In rs7251, CG genotype may increase the CLL risk. In the rs2304206, the alleles T may increase the CLL risk. The GTC haplotype can decrease the CLL risk in normal people, the GTT haplotype can increase the CLL risk in normal people. After treatment, in the rs7251, the event-free survival (EFS) in patients carrying CC genotype was higher than those carrying CG + GG genotype. In the rs2304206, the EFS in patients carrying CC genotype was higher than those carrying CT + TT genotype. IRF-3 gene polymorphisms were associated with the susceptibility and prognosis of CLL, it can be used as an auxiliary index for clinical detection of CLL.

Exploration of molecular mechanisms of diffuse large B-cell lymphoma development using a microarray.

OBJECTIVE: We aimed to identify key genes, pathways and function modules in the development of diffuse large B-cell lymphoma (DLBCL) with microarray data and interaction network analysis. METHODS: Microarray data sets for 7 DLBCL samples and 7 normal controls was downloaded from the Gene Expression Omnibus (GEO) database and differentially expressed genes (DEGs) were identified with Student's t-test. KEGG functional enrichment analysis was performed to uncover their biological functions. Three global networks were established for immune system, signaling molecules and interactions and cancer genes. The DEGs were compared with the networks to observe their distributions and determine important key genes, pathways and modules. RESULTS: A total of 945 DEGs were obtained, 272 up-regulated and 673 down-regulated. KEGG analysis revealed that two groups of pathways were significantly enriched: immune function and signaling molecules and interactions. Following interaction network analysis further confirmed the association of DEGs in immune system, signaling molecules and interactions and cancer genes. CONCLUSIONS: Our study could systemically characterize gene expression changes in DLBCL with microarray technology. A range of key genes, pathways and function modules were revealed. Utility in diagnosis and treatment may be expected with further focused research.

[Detection and identification of seven clinical common pathogenic bacteria by oligonucleotide microarray].

OBJECTIVE: Using 16S rDNA and 23S rDNA genes as the target sequences to develop a system based on oligonucleotide microarray and to detect the seven clinical pathogenic bacteria, commonly seen. METHODS: Double polymerase chain reaction (PCR) was applied to amplify the segments of 16S rDNA and 23S rDNA genes of the target bacteria. An oligonucleotide microarray was constructed to simultaneously detect EHEC O157:H7, Vibrio parahaemolyticus, Salmonella sp., Vibrio cholerae, Listeria monocytogenes, Campylobacter jejuni and Shigella sp. Specificity, sensitivity and reproducibility of the microarray during detection were checked. And then microarray was used to detect the microbes in stool specimens of 81 patients with diarrhea and vomiting. RESULTS: The double PCR method could simultaneously amplify the target sequences of 16S rDNA and 23S rDNA genes of the seven pathogens. The sensitivity of the developed oligonucleotide microarray could reach 10(3) cfu/ml but no positive results were presented for non-targeted bacteria. The coefficients of differentiation in one lot or among different lots of the microarray slices were 3.89% - 5.81%. The positive detection rate of the stool specimens by oligonucleotide microarray was 39.5% (32/81), with a coincidence of 96.3% (78/81) for the patients and another coincidence of 96.8% (31/32) for bacterial genus or species identification, when comparing to the results by routine bacteriological examinations. CONCLUSION: The established assay in this study based on oligonucleotide microarray to detect the seven pathogenic bacteria has many advantages such as convenient, rapid, accurate, stable and high flux, which is suitable for clinical specimen examination and epidemiological field investigation.

[Detection and identification of human metapneumovirus by real time reverse transcription PCR].

OBJECTIVE: To develop a rapid, sensitive and specific real time reverse transcription PCR for detecting and identifying human metapneumovirus. METHODS: The Hmpv-L gene of human metapneumovirus was chosen as target gene, the primers and TaqMan probe were designed, and the PCR reaction was optimized systematically. The total RNA was extracted from respiratory specimens, and reverse transcription was performed through random primer. The cDNA was detected by using real time PCR. The specificity, sensitivity and reproducibility of real time PCR were estimated. The real time PCR was applied to detect 180 clinical respiratory specimens. RESULTS: The human metapneumovirus can be detected using real time reverse transcription PCR accurately and quickly, and the sensitivity was 1 copy/microl. The coefficient of variation of intra-assay and inter-assay was less than 5%. Among those 180 specimens, 28 (15.56%) were positive for human metapneumovirus, the clinical diagnoses for these 28 patients were pneumonia (15.60%, 17/109) and bronchiolitis (15.49%, 11/71). 21 positive specimens were from patients under 2 years of age, and 6 positive specimens were from patients between 2 and 5 years of age, only 1 positive specimens was from patients over 5 years. CONCLUSION: It is demonstrated that real time reverse transcription PCR is a reliable, accurate and feasible assay for human metapneumovirus, which has become one of the most important pathogens induced acute respiratory infections in pediatric patients.