Method for Solving Non-specific Amplification Interference of Fluorescence Quantitative PCR in Gene Detection

Objective:To explore a method to solve the issue of interference in fluorescence quantitative PCR non-specific amplification for gene detection.Method:A three-step method was used for amplification,and the quantitative fluorescence signal collection process was set in the extension stage.Results:Three-step amplification has the advantages of wide application range;improved accuracy;and reduced primer design requirements.Conclusion:The interference of non-specific amplification signals was effectively avoided,the melting curve plotting process was omitted,the reaction time was shortened,and the detection accuracy was improved.

Establishment of a Real-time Fluorescent Quantitative PCR Assay for Detection of Genetically Modified Maize Line MON88017

In order to improve the standardized technical systems of quantitative analyses for genetically modified organisms(GMOs) and products,ensure bio-safety and reduce ecological risk in China,a real-time fluorescent quantitative PCR assay was established for detection of genetically modified maize line MON88017.The established method was evaluated based on the specificity,sensitivity,accuracy and measurement uncertainty.The results showed that the established method had strong specificity in detection of genetically modified maize line MON88017.1.50%MON88017 sample was detected with 29 replications.The average measured value(1.541%) was close to the actual value(1.50%) and the relative deviation was 2.70%.The variation coefficient of the measured value was 0.110 4;the recovery was 100.00%and the measurement uncertainty was 0.096.The limit of detection for genetically modified maize line MON88017 with the established method was 5 copies at the 97.5%confidence level.Thus,the real-time fluorescent quantitative PCR assay established in this study exhibited high specificity,accuracy and sensitivity,which could provide technical support for the safety supervision of genetically modified organisms and products in China.

Expressions of Toll Like Receptor(TLR)Genes in Paralichthys olivaceus After Induced by Different Extracts of Edwardsiella tarda

Toll like receptors(TLRs)are the main innate immune‘pattern recognition receptors’of animals,which play a central role in host cell recognition and responses to invasive pathogens,particularly common structures of microbial pathogens.In this study,the gene expression profiles of TLRs in the spleen,head kidney,gill,small intestine,liver,muscle,and heart of healthy Paralichthys olivaceus were detected by real-time quantitative PCR(qPCR).The TLR family members were widely expressed in different tissues with different basic expression profiles.The highest expressions of TLR1,5m,7,8,9,14,and 21 were found in the spleen;the highest expressions of TLR3 and TLR21 were found in the gill;the highest expressions of TLR2 and 5s were found in the small intestine.The second highest expressions of TLR3,7,and 8 were found in small intestine.The gene expression profiles of TLRs stimulated with Edwardsiella tarda DNA,RNA,and lipopolysaccharide(LPS)were also detected in spleen,head kidney and gill.TLR9 and TLR21 were sensitive to E.tarda DNA;TLR 8 and TLR21 were sensitive to E.tarda RNA;and TLR1 and TLR14 were sensitive to E.tarda LPS.The expressions of the other TLR genes showed no significant changes.The results imply that the expressions of these TLR genes in P.olivaceus are differently regulated in the whole body and play important roles in the immune response against E.tarda infection.

Screening for Enterobacteriaceae Bacteria in Infant Formula Powder

A total of 151 infant formula powder samples were tested for the screening of the Enterobacteriaceae bacteria, and it had been contaminated by the conditioned pathogen like Enterobacter sakazakii, Klebsiella pneumoniae, Klebsiella oxytoca, and Enterobacter cloacae. The results showed that six samples were separated Enterobacter sakazakii, 24 samples were separated Enterobacteriaceae by using the traditional validation and real-time fluorescence quantitative PCR. Then the result identified by API20E biochemistry test and VITEC2 identification system. Through the study we concluded the potential safety hazard of the current infant formula powder and should regard Enterobacteriaceae as an indicator to carry out effective management, the neglected mild and discontinuity contamination could be easily found, thus to guarantee the safety of the special group.

Transformation of ammonium nitrogen and response characteristics of nitrifying functional genes in tannery sludge contaminated soil

High concentrations of ammonium nitrogen released from tannery sludge during storage in open air may cause nitrogen pollution to soil and groundwater.To study the transformation mechanism of NH_(4)^(+)-N by nitrifying functional bacteria in tannery sludge contaminated soils,a series of contaminated soil culture experiments were conducted in this study.The contents of ammonium nitrogen(as NH_(4)^(+)-N),nitrite nitrogen(as NO_(2)^(−)-N)and nitrate nitrogen(as NO_(3)^(−)-N)were analyzed during the culture period under different conditions of pollution load,soil particle and redox environment.Sigmodial equation was used to interpret the change of NO_(3)^(−)-N with time in contaminated soils.The abundance variations of nitrifying functional genes(amoA and nxrA)were also detected using the real-time quantitative fluorescence PCR method.The results show that the nitrification of NH_(4)^(+)-N was aggravated in the contaminated silt soil and fine sand under the condition of lower pollution load,finer particle size and more oxidizing environment.The sigmodial equation well fitted the dynamic accumulation curve of the NO_(3)^(−)-N content in the tannery sludge contaminated soils.The Cr(III)content increased with increasing pollution load,which inhibited the reproduction and activity of nitrifying bacteria in the soils,especially in coarse-grained soil.The accumulation of NO_(2)^(−)-N contents became more obvious with the increase of pollution load in the fine sand,and only 41.5%of the NH_(4)^(+)-N was transformed to NO_(3)^(−)-N.The redox environment was the main factor affecting nitrification process in the soil.Compared to the aerobic soil environment,the transformation of NH_(4)^(+)-N was significantly inhibited under anaerobic incubation condition,and the NO_(3)^(−)-N contents decreased by 37.2%,61.9%and 91.9%under low,medium and high pollution loads,respectively.Nitrification was stronger in the silt soil since its copy number of amoA and nxrA genes was two times larger than that of fine sand.Moreover,the copy numbers of amoA and nxrA genes in the silt soil under the aerobic environment were 2.7 times and 2.2 times larger than those in the anaerobic environment.The abundance changes of the amoA and nxrA functional genes have a positive correlation with the nitrification intensity in the tannery sludge-contaminated soil.