Methodological study on eliminating nucleic acid contamination in molecular diagnostic laboratory / 生物工程学报

Nucleic acid detection technique has good sensitivity and specificity and is widely used in in vitro diagnosis, animal and plant commodity quarantine, forensic identification, and other fields. However, it is susceptible to carryover contamination during the operation and leads to false-positive results, which seriously affects the detection accuracy. Therefore, finding an effective solution to prevent and eliminate nucleic acid carryover contamination has become particularly urgent. This study compared several different methods for removing nucleic acid contamination and confirmed that sodium hypochlorite solution and PCRguard reagent could effectively eliminate nucleic acid carryover in the liquid and on surfaces of different materials. Besides, the combination of sodium hypochlorite solution and PCRguard can solve the nucleic acid aerosol contamination. This study proposes solutions for the routine prevention of carryover contamination and removal of aerosol that has occurred in molecular diagnostic laboratories.

Development and characterization of serotype-specific monoclonal antibodies against Dengue virus NS1 / 生物工程学报

Dengue virus (DENV) is the most widely transmitted arbovirus in the world. Due to the lack of diagnostic technology to quickly identify the virus serotypes in patients, severe dengue hemorrhagic fever cases caused by repeated infections remain high. To realize the rapid differential diagnosis of different serotypes of DENV infection by immunological methods, in this study, four DENV serotype NS1 proteins were expressed and purified in mammalian cells. Monoclonal antibodies (MAbs) against NS1 protein were obtained by hybridoma technology after immunizing BALB/c mice. Enzyme-linked immunosorbent assay, indirect immunofluorescence assay, dot blotting, and Western blotting were used to confirm the reactivity of MAbs to viral native NS1 and recombinant NS1 protein. These MAbs include not only the universal antibodies that recognize all DENV 1-4 serotype NS1, but also serotype-specific antibodies against DENV-1, DENV-2 and DENV-4. Double antibody sandwich ELISA was established based on these antibodies, which can be used to achieve rapid differential diagnosis of serotypes of DENV infection. Preparation of DENV serotype-specific MAbs and establishment of an ELISA technology for identifying DENV serotypes has laid the foundation for the rapid diagnosis of DENV clinical infection.

A rapid and quantitative fluorescent microsphere immunochromatographic strip test for detection of antibodies to porcine reproductive and respiratory syndrome virus

A fluorescent microsphere-based immunochromatographic strip test (FICT) was developed for the rapid, sensitive, and quantitative detection of porcine reproductive and respiratory syndrome virus (PRRSV) antibodies at the pen-side. The assay was based on the formation of a sandwich immune-complex (anti-pig IgG-PRRSV antibodies-NSP7/N), which was validated by a comparison with IDEXX-ELISA using 3325 clinical specimens. The diagnostic specificity, sensitivity, and accuracy of FICT were 97.28, 93.41, and 94.95%, respectively. FICT showed a good correlation with the virus neutralization assay. Overall, a promising pen-side diagnostic tool was developed for the rapid and quantitative detection of PRRSV antibodies within 15 min.

A novel carbon nanoparticle probe-based ultrasensitive lateral flow assay for rapid detection of Ebola virus / 生物工程学报

Ebola virus (EBOV) is an extremely contagious pathogen first discovered in Africa associated with severe hemorrhagic disease in humans and nonhuman primates, which has resulted in at least 28 500 suspected cases and 11 300 confirmed deaths in 2014-2016 Ebola epidemic in West Africa. Rapid and sensitive detection of EBOV is the key to increasing the probability of survival and reducing infection rates in pandemic regions. Here, we report an ultrasensitive and instrument-free EBOV detection assay based on colloidal carbon immunochromatography. Carbon nanoparticle-labeled rabbit anti-EBOV-VP40 IgG were concentrated in the conjugate pad, monoclonal antibody (McAb, 4B7F9) against EBOV-VP40 and goat anti-rabbit IgG were immobilized on the nitrocellulose membrane with 2 μL/cm at a concentration of 1 mg/mL as test and control lines, respectively. Then the sample application pad, conjugate release pad, nitrocellulose membrane and absorbent pad were assembled into a lateral flow test strip. The test strip shows strong specificity against related viruses that share similar clinical symptoms and geographic range with EBOV, including marburg virus, influenza virus, yellow fever virus and dengue virus. In addition, 1 500 negative serums were tested with false-positive rate of 1.3‰ which significantly lower than that of ReEBOV™ colloidal gold test kit recommended by World Health Organization (WHO). The sensitivity of this strip was analyzed using inactivated EBOV with detection limit of 100 ng/mL (10⁶ copies/mL) which clearly higher than that of ReEBOV™ dipstick (10⁸ copies/mL). Furthermore, the strip showed excellent thermal stability characteristics in room temperature and could be as a point-of-care (POC), ultra-sensitive and specific promising candidate for EBOV serological screening in rural Africa or entry/exit ports.

Thyroid functional changes of normal human fetus and newborns / 中华妇产科杂志

Objective To study the changes of the thyroid hormones leval of human fetus and newborans Methods More than 71 cases of medically indicated cordocentesis have been done in 16 36 gestational weeks in our hospital during last three years. Among them, 71 fetus who were free of diseases and their maternal thyroid function were normal were included into the study group. The blood samples were sent to analysis of thyroxine (T 4), triiodothyroxine (T 3), free thyroxine (FT 4), free triiodothyroxine (FT 3) and thyrotropin (TSH). 140 umbilical cord blood samples taken at the time of term delivery were sent to analysis of FT 4,FT 3 and TSH as a control. Normal range of different gestational weeks was calculated. Statistical analysis was done for the changes of all these thyroid hormones before 28 weeks and after Results All the thyroid hormones can be detected in 16 weeks of pregnancy,FT 4 already reaches the top level of adults (5.8?2.6) pmol/L and will continually increase with the increase of gestational age. There was a parallel increment of all the fetal thyroid hormone concentrations with the gestational age. The concentrations of T 4,T 3 and FT 4 have a rapidly increase after 28 weeks and have a statistically significant difference from (2.8 ?1.8) nmol/L, (37.2?27.2) nmol/L and (10.6?3.1) pmol/L,respectively to (5.8?2.6) nmol/L, (55.9?33.3) nmol/L,( 13.0?4.5) pmol/L, respectively. TSH level of fetus was increased gradually along the gestation, reaching the up level of the adults at the 20 weeks and peaking at the birth time. While the T 3 and FT 3 keep in a lower level in gestation Conclusions Fetal thyroid hormones increase with the gestational age.The diagnosis of congenital fetal thyroid hormone malfunction in the second half of the pregnancy should be monitored mainly by the T 4, FT 4 and TSH levels in different gestational age. For this consideration, to set up a reliable data for normal human fetus thyroid hormone concentrations is a very important and essential step to provide a practical guide for doctors to do intra uterine diagnosis and treatment of associated high risk groups. The peaking level of TSH at the birth time will surely company the changing of other thyroid hormones, so it might not be the best time to screening the congenital thyroid malfunction at the 72 hours after birth.

Method and Complications of Direct Fetal Blood Sampling / 中华围产医学杂志

Objective To study the indications of percutaneous ultrasound-guided direct fetal blood sampling; the methods of fetal blood assessment; and the relationship between the different puncture techniques and the complications. Methods Direct fetal blood sampling were performed on 90 pregnant women whose gestational age were from 16 to 36 weeks for medical indications under the guide of ultrasound. None stress test were performed before and after the puncture. Ulrtrasonographies were done at 2 hours and 24 hours after the procedure. Results There were no severe maternal or fetal complications such as fetal loss and placental abruption. The incidence of bradycardia was 35.6%(32/90), all recovered within 60 seconds; the incidence of bradycardia happened within 4 hours after the puncture was 2.25(2/90). There was a positive relationship among the incidence of bradycardia, the volume of blood taken and the place of punctures,r=0.27 and 0.36 respectively. There was one case with placental changing happened after 2 hours of the puncture, the placenta changing incidence was 1.1%. No emergency cesarean section was done due to the complications of the procedure. All the umbilical cord were normal when the babies were born. Conclusions Direct fetal blood sampling is a safe technique for both fetus and the mother under the guide of real time ultrasound. The data come from fetal blood analysis give reliable informations about chromosome, congenital infections, the status of fetal metabolism, endocrine system and hematologic system directly, which make this technique unreplacable for intrauterine diagnosis.