| Armored RNA technology is a mature technology that may be well suited for preparation of RNA-based control sample or standard material.Armored RNA technology works by inserting sequences of bacteriophage MS2 coated protain and exogenous gene into multiple clone site(MCS)of expression vector by genetic engineering approach,and then expresses armored RNA,or termed MS2 virus-like particles(VLPs),with RNA packaged inner the coated protain of MS2.Under the protection of MS2 coated protain,the inner RNA can keep stable without degradation by RNA ribonucleases;meanwhile,with similar structure to real virus,the armored RNA can be used to prepare reagent,such as control sample,standard material,for diagonosis of diseases,especially for infectious diseases.In our previous studies,we have prepared several kinds of armored RNAs for detecting Influenza,Ebola,Middle East respiratory syndrome(MERS),et al.,and organized external quality assessment(EQA)on these assays in China.Dengue is an acute infectious disease,transmited by aedes aegypti,which is also the most widely spreading disease in the world,mainly in tropical and subtropical area.In 2014,dengue epidemic of Guangzhou in China was caused by imported cases,and then spread to 20 other cities and other provinces,resulting in more than 46 thousands of cases in China;during 2015 and 2017,several thousands of infectious cases also reported in China.Chronic myeloid leukemia(CML)and acute lymphocytic leukemia(ALL)are two common leukemias in the world.Majority of CML and part of ALL are characterized by Philadelphia chromosome(Ph).The Philadelphia chromosome(Ph)and the resultant BCR-ABL1 fusion gene(FG)resulted from reciprocal translocation between the ABL-1 oncogene of chromosome 9 and a breakpoint cluster region(BCR)on chromosome 22,termed t(9;22)(q34;q11).CML is commom with p210 BCR-ABL1 fusion gene(FG),while p190 BCR-ABL1 FG subtype often occurs in Ph positive ALL(Ph+ ALL).Reverse transcription-quantitative PCR(RT-qPCR)is a routine and sensitive approach,which can be used to diagnose dengue infection and leukemia,and is widely used in majority of laboratories in China.Due to complex operation,RT-qPCR can be affected by several factors,leading to unstable results between laboratories.EQA is an effective tool to compare results between laboratories and improve laboratory diagnostic capacities;however,no EQA schemes,no matter dengue or leukemia,have been organized in China before.In this,we prepared control samples for dengue(Part I)and BCR-ABL1FG transcript(PartⅡ)detection using armored RNA technology.In part Ⅰ,we prepared dengue 1-4 control samples,which contain corresponding segments of dengue 1～4 for RT-qPCR.Each panel contains 5 positive samples and 3 nagative ones.Then dengue 1～4 sample panels were sent to 20 participanting laboratories in China.The result showed that the accuracy,sensitivity,and specificity of the 20 evaluated laboratories were 89.6%(569/635),85.1%(336/395),and 97.1%(233/240),respectively.Overall,sixteen(80.0%)laboratories were qualified in detection of dengue virus,among which five(25.0%)laboratories were designated as "competent",eleven laboratories(55.0%)were designated as "acceptable’,whereas four laboratories(20%)were designated as"improvable".In part Ⅱ,we prepared p210 and p 190 BCR-ABL1 control samples by mixing variable p210 and p190 BCR-ABL1 FG armored RNAs to constant CG armored RNAs.Each sample panel contains nine positive sampes,among which one sample was defined as the benchmark samples for calculating Z scores of other samples,and one negative sample.The result show that CV%of%BCR-ABL results,no matter p210 subtype or p190 subtype,basically exceeded 60%,even 100.0%or 200.0%.Overall,CV%of p210 subtype was lower than p190 subtype.In 24 international scale(IS)laboratories,the CV%of results of p210 subtype decreased from 82.4%to 61.6%after conversion with a laboratory-specific conversion factor(CF);however,poorly converted results were also observed in 3 laboratories,among which 2 laboratories changed their components of RT-qPCR procedure recently.The result from part Ⅰ and part Ⅱ indicated that in detection of dengue virus and BCR-ABL FG transcripts by RT-qPCR,all kinds of problems were found in local laboratories in China.By solving the existing problems,the performance of dengue virus and BCR-ABL FG transcripts detection can be improved,ensuring robust laboratory diagnostic capacities in China.Using universal standard can improve comparability of results from different laboratories;however,the widely used standards in laboratories are basically plasmid or cDNA,which can not reflect the entire procedure of RNA extraction and cDNA synthesis.In part Ⅲ,we prepared universal p210 and p190 BCR-ABL 1 standards(p210FG-CG,p190FG-CG)based on armored RNA technology.To decrease variability of BCR-ABL 1 detection due to two standards of BCR-ABL 1 and CG,we linked p210 or p190 BCR-ABL1 FG transcript with four CGs(ABL1,BCR,GUSB,and B2M)at a ratio of 1:1.The result showed that when using local standard,p210 and p190 samples with CV%higher than 80.0%accounted for 33.3%(3/9)and 55.6%(5/9),respectively;samples with CV%between 50%-80.0%accounted for 66.7%(6/9)and 44.4%(4/9),respectively;while no simulated sample with CV%lower than 50.0%.By contrast,when using p210FG-CG and p190FG-CG standard,samples with CV%higher than 80.0%,between 50%～80.0%,and lower than 50.0%all accounted for 33.3%(3/9).We also found that CV%of simulated sample results decreased more significantly in laboratories using spin column method to extract RNA and using laboratory developed tests(LDTs).The study of part Ⅲ indicates that p210FG-CG and p190FG-CG prepared here can improve the comparability of BCR-ABL 1 detection results between laboratories.Although using universal standard can improve comparability of results from different laboratories,it is important to note that the use of universal standard does not by itself produce results on the international scale(IS).Two ways are suggested for conducting standardization of p210 BCR-ABL1 FG transcript quantification,sample exchange and WHO 1st Reference Panel for quantitation of BCR-ABL mRNA.Although the way of sample exchange works well for laboratories with tests that show good stability over time,it is time consuming,expensive and difficult to access for smaller laboratories;meanwhile,restricted access to the WHO1st panel,which is currently only available to manufacturers of BCR-ABL1 test kits and secondary standards.Additionally,the standardization of p190 BCR-ABL1 subtype has not been established.So it is essential to develop the second reference material tracing to the WHO1st panel.In part IV,we developed armored RNA-based second reference material for standardization of p210 and p190 BCR-ABL1 quantification.To ensure the amplification segments of p210 and p190 BCR-ABL1 stay the same,we replace part(75bp)of sequence of p190 BCR-ABL1 exon 1 with p210 BCR-ABL1 exon 14(75bp)using OE-PCR.The value assignment of the 4 levels of the second reference materials were conducted following the suggested method for calibration of secondary standards from WHO1st panel.The second reference materials were also replenished with 23s RNA as exogenous RNA and were freeze-dried before they were sent to the participanting laboratories.The result showed that in the 33 participanting laboratories,75.8%(25/33)laboratories obtained valid CFs for p210 BCR-ABL1 assay,and 84.8%(28/33)laboratories obtained valid CFs for p190 BCR-ABL1 assay.Meanwhile,among the 15 IS laboratories whose CF obtained were valid,80.0%(12/15)achieved a CF ratio between 0.63 and 1.58,indicating that their current CF was equivalent to the CF from the secondary panel.In sum,we successfully prepared dengue and p210/p190 BCR-ABL1 control samples,p210/p190 BCR-ABLI standard and second reference materials using armored RNA technology.The study is helpful in finding the existed problems in dengue and BCR-ABL1 FG detection,which is hepful to improve the comparability and standardization of BCR-ABL1 FG quantification,so as to ensure robust laboratory diagnostic capacities in China. |
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