Cuantificación de la expresión del gen amo-A en poblaciones bacterianas y archaeales presentes en muestras de suelos de un lote arrocero caracterizado por ambientes

El ciclo del nitrógeno representa uno de los procesos biogeoquímicos más importantes para los ecosistemas terrestres y acuáticos. Las comunidades microbianas desempeñan un papel crucial en los procesos de transformación del nitrógeno en el suelo, ya que participan en diversas etapas como la nitrificación, de gran importancia para la producción agrícola. Dentro de los marcadores moleculares más utilizados para evaluar la actividad de poblaciones microbianas oxidantes de amonio se han considerado ampliamente los genes que codifican enzimas claves como la subunidad A de la actividad amonio monooxigenasa (AMO). Sin embargo, no se comprende completamente si la expresión de esta enzima tiene relación directa con el rendimiento de los cultivos. En este contexto, se evaluó la expresión del gen amo-A de comunidades bacterianas y archaeales presentes en un lote arrocero previamente caracterizado por ambientes. Para cuantificar la abundancia de arqueas y bacterias oxidantes de amonio, (AOA y AOB, respectivamente) se emplearon las técnicas de PCR en tiempo real (RT-qPCR) y PCR digital (RT-dPCR). En este trabajo se encontró a través del análisis de datos metagenómicos que hubo una mayor presencia de AOB en las muestras de suelo rizosférico mientras que las AOA fueron predominantes en las muestras de suelo de soporte "bulk", sin embargo, no se detectó la expresión del gen amo-A asociada a la comunidad de bacterias en las muestras de suelo analizadas. Por otra parte, no se presentaron diferencias entre los transcritos del gen amo-A asociados a la comunidad de AOA de los ambientes caracterizados. Además, la expresión de transcritos no estuvo relacionada con alguna de las propiedades químicas evaluadas. Finalmente, las estrategias de cuantificación para RT-qPCR (plásmido y templete) resultaron ser homólogas y funcionales para identificar la expresión del gen amo-A de AOA, mientras que la técnica de RT-dPCR fue más precisa para el análisis de la comunidad de AOB y AOA.

The nitrogen cycle represents one the most important biogeochemical process for terrestrial and aquatic ecosystems. Microbial communities play a crucial role in the processes of transformation of soil nitrogen in the, since they participate in various stages such as nitrification, which is of great importance for agricultural production. Among the most used molecular markers to assess ammonium oxidizing microbial populations activity have been considered widely the genes encoding key enzymes such as ammonium monooxygenase (AMO) subunit A. However, it is not fully understood whether the expression of this enzyme is directly related to the crop yield. In this context, this research work evaluated the expression of the amo-A gene of bacterial and archaeal communities present in a rice field previously characterized by environments. Real-time PCR (RT-qPCR) and digital PCR (RT-dPCR) techniques were used to quantify the abundance of archaea and ammonium-oxidizing bacteria (AOA and AOB, respectively). In this work it was found that in the analysis of metagenomic data there was a greater presence of AOB in rhizospheric soil samples while AOA were predominant in bulk soil samples, however, the expression of the amo-A gene was not detected. associated with the community of bacteria in the soil samples analyzed. On the other hand, it was found that the transcripts of the amo-A gene of the AOA community did not present differences between the characterized environments. Furthermore, the expression of transcripts is not related to any of the chemical properties evaluated. Finally, the quantification strategies for RT-qPCR (plasmid and quenching) turned out to be homologous and functional to identify the expression of the AOA amo-A gene, while the RT-dPCR technique was more precise for the analysis of the community of AOB and AOA.

Evaluation of stability of Luc2P reporter cell lines by duplex digital PCR / 中国生物制品学杂志

@#Objective To develop a duplex digital PCR(dPCR) for evaluation of the stability of luciferase(Luc) gene in Luc2P reporter cell lines(CHO-K1,Hacat,HEK293 and UT7).Methods Genomic DNAs of Luc2P reporter cell lines were extracted,a duplex dPCR was developed to determine the copies of reference gene RPP30 and the target gene Luc,and the relative copy number of Luc(copies Luc/copy RPP30) was employed as the indicator for the stability evaluation of Luc gene;The developed method was verified for the specificity,precision,linearity,accuracy and durability,and analyzed for the applicability,according to the related requirements in Chinese Pharmacopoeia(Volume Ⅲ,2020 edition).Results All the original cells without reporter gene transfection were negative,while all the four reporter cell lines were positive,and the negative and positive regions in dPCR results were clearly distinguished;The relative standard deviation(RSD) of the eight repeated detections of the same genomic DNA sample and the six independent extractions of genomic DNA sample of the same cell were all less than 10%,and the linear fitting R~2 values were more than 0.99 for both Luc and RPP30.The spike recoveries of five groups of samples detected by the developed method were between 50% and 100%,and the results of chip-type dPCR and droplet-type dPCR were consistent.This method distinguished the relative copy number of Luc in different cell clones,and the results of detecting the relative copy number of Luc in three passages(P8,P12 and P31) were highly consistent.Conclusion The developed duplex dPCR method has good specificity,precision,linearity,accuracy,durability and applicability,and might be used to evaluate the stability of Luc gene in Luc2P reporter cell lines.

Quantification of cell-free circulating mitochondrial DNA copy number variation in hepatocellular carcinoma

SUMMARY OBJECTIVE: Hepatocellular carcinoma is the most common primary malignant liver tumor. Mitochondrial DNA copy number has been shown to be associated with various malignancies. However, there has not been any study on the absolute quantification of mtDNA copy number in hepatocellular carcinoma. The aim of this study was to develop a new method for absolute quantification of mtDNA copy number and to relatively quantify the variations in the mtDNA copy number in hepatocellular carcinoma patients in comparison with healthy individuals. METHODS: Venous blood samples were collected from both hepatocellular carcinoma patients (34) and healthy individuals (34). Circulating cell-free DNAs were isolated and the relative quantification of mtDNA copy number variation was determined using quantitative polymerase chain reaction and digital polymerase chain reaction. RESULTS: It was found that the relative mtDNA copy number was significantly decreased in hepatocellular carcinoma patients in comparison with the control group (p<0.05). The median (range) and average of relative mtDNA/β-actin gene of the patients were determined as 42.8 cp/μL (11.1-88.5) and 45.1 cp/μL, respectively, while the median (range) and average relative mtDNA/β-actin gene of the control group were determined as 102.8 cp/μL (55.1-291.8) and 138.7 cp/μL, respectively (p<0.05). When quantitative polymerase chain reaction and digital polymerase chain reaction were compared, mtDNA/β-actin gene copy number ratio of digital polymerase chain reaction results was found to be 1.76-fold more than that of quantitative polymerase chain reaction results. CONCLUSION: Circulating mtDNA copy number was decreased in hepatocellular carcinoma patients in comparison with healthy individuals, and we suggest that it can be used as a noninvasive biomarker for hepatocellular carcinoma diagnosis in the future.

Fluorescence PCR Amplification Related Technology / 中国生物化学与分子生物学报

Using fluorescence PCR (FPCR) technology to amplify DNA is an important part of modern biological research. The paper traced the invention of FPCR, through its main development, respective principles, design techniques, through to practical applications, etc. The two generations of phased methods of real-time quantitative PCR (QPCR) and digital PCR (DPCR) were mainly reviewed. QPCR that contained means of dyes, hydrolysis probe and its derivatives, hybridization probe containing molecular beacon and Yin-Yang probes, etc, dye melting curve and probe melting curve was summarized. DPCR involving chip digital PCR(cdPCR) and droplet digital PCR (ddPCR) was also included. Furthermore, the main application areas and limitation of FPCR, their characteristics of different types and future development direction were described.