| As RNA research continues to progress,people’s knowledge of RNA types and functions is increasingly enriched,and the demand for RNA detection in clinical and scientific research is increasing.RNA viruses cause a variety of epidemic diseases,which have a huge impact on public health and personal health.Timely diagnosis of pathogens is helpful to cut off the transmission route and curb the spread of diseases;monitoring the load of virus carriers is helpful to judge the disease progression,guide clinical medication,and observe the efficacy and prognostic effect.With the increasing incidence of cancer,some new therapeutic modalities such as targeted therapy and immunotherapy have been applied to give cancer patients new hope for life,and the expression products of certain fusion genes(m RNA)have become markers for some tumors.For the detection of appeal RNA,simple and rapid assays with high sensitivity and specificity are needed.General RNA detection requires conversion of RNA to DNA,including reverse transcriptase-based and ligase-based detection methods.However,the lack of single-strand DNA ligation activity of most DNA ligases using RNA as template limited the use of ligasebased assays in RNA detection until the discovery of SplintR ligase with high efficiency of single-strand DNA ligation using RNA as template,and the discovery of SplintR ligase opened up the prospect of using ligase-based assays in RNA detection such as mi RNA and circular RNA.In this study,we established a qualitative and quantitative RNA detection method based on SplintR ligase,including reaction system establishment and methodological validation,using viral genomic RNA and m RNA of tumor-associated fusion genes as targets.First: viral RNA detection based on SplintR ligase mainly consists of two parts: ligation reaction and ligation product detection.The ligation product detection methods include fluorescent quantitative PCR(q PCR),polyacrylamide gel electrophoresis(PAGE),and capillary electrophoresis(CE)detection techniques.RNA was first obtained by constructing hepatitis C virus(HCV)and human immunodeficiency virus(HIV)pseudoviruses and in vitro transcription as RNA standards for subsequent assays.The ligation reaction system was established by optimizing the annealing conditions of hybridization,ligase volume,ligation reaction temperature,and ligation probe concentration;optimizing the primer probe concentration to establish the multiplex fluorescence quantitative PCR detection reaction system;optimizing the annealing temperature,ligation probe and primer concentration to establish the capillary electrophoresis detection system.The sensitivity,specificity,precision and anti-interference ability of the established system were then verified.SplintR ligase based single q PCR method was able to detect 100 copies/m L of HIV pseudovirus,1000 copies/m L of HCV pseudovirus,100 copies/μL of H1N1 virus RNA,10 copies/μL of H5N1 virus RNA;SplintR ligase based dual q PCR method was able to detect 100 copies/μL H1N1 and H5N1.SplintR ligase based triple q PCR method was able to detect 100 copies/m L of HIV and 100copies/μL of H1N1 and H5N1 RNA.The coefficient of variation of the precision assay was less than 1%.SplintR ligase based single PAGE assay was able to detect 10 copies/m L of HIV and100 copies/m L of HCV;SplintR ligase based dual PAGE assay was able to detect 10 copies/m L of HIV and 10 copies/m L of HCV;the CE method based on multiplex SplintR ligase was able to simultaneously detect 1000 copies/m L of HIV,HCV and 1000 copies/μL of H1N1,H5N1 RNA viruses.Second: A reverse transcription-based multiplex q PCR and SplintR ligase-based q PCR protocol was established to detect the EML4-ALK(microtubule-associated protein-like 4-mesenchymal lymphoma kinase)fusion gene.The length and concentration of ligation probe used for the ligation reaction were optimized;the primer and probes for the multiplex quantification system were optimized.The sensitivity,specificity,precision and interference resistance were also verified.Detection of EML4-ALK fusion gene RNA abundance by SplintR ligase-based q PCR;detection of clinical lung cancer samples by two methods.The SplintR ligase-based q PCR method could detect 10copies/μL of variants 1 and 2 of the EML4-ALK fusion gene and 100copies/μL of variant 3a/b of the EML4-ALK fusion gene,and determined the mutation abundance of EML4-ALK variant 1 positive samples,variant 2 positive samples as 0.514±0.006,0.633±0.010.The reverse transcription-based multiplex q PCR method could detect 10copies/μL of EML4-ALK fusion gene for variants 1 and 3a,1 copies/μL of EML4-ALK fusion gene for variant 2,and 100 copies/μL of EML4-ALK fusion gene for variant 3b.Both ligase-based q PCR and reverse transcription-based multiplex q PCR assays detected 9cases of EML4-ALK positivity in 57 clinical lung cancer samples.In this paper,we established SplintR ligase based multiplex detection protocols for HCV,HIV,influenza A virus H1N1 and H5N1 and q PCR based EML4-ALK fusion typing detection protocols.q PCR technology based on SplintR ligase is expected to show a promising future in RNA detection. |
