Application Of Reference Materials In Detection Of Encephalitis-related RNA Viruses And Non-invasive Prenatal Testing Of Fetal Microdeletions And Microduplications Syndrome

Detection of encephalitis-related viruses and non-invasive prenatal testing(NIPT)of fetal microdeletion/microduplication syndrome are two routinely tests in clinical molecular diagnostic laboratories.For the detection of encephalitis-related viruses,extracting RNA with sufficient concentration from cerebrospinal fluid with low viral load is a challenge.For fetal microdeletion/microduplication NIPT,the experimental analysis process is numerous and complex,including sampling,transportation and preservation,cell-free DNA(cfDNA)extraction,library construction,next generation sequencing(NGS),bioinformatics analysis and other steps,and the experimental operation has a great impact on the detection results.Therefore,these two testing are in urgent need of a reliable quality control product to carry out indoor quality control and external quality assessment(EQA).In this study,the reference materials of the above two routine detection methods are studied respectively.Nine encephalitis-related RNA viruses detection and five fetal microdeletion/microduplication syndromes NIPT reference materials were prepared by VLP technology and enzyme-digested nuclear technology,respectively.EQA were carried out in laboratories across the country to comprehensively assess the overall testing ability of laboratories.Meanwhile,the daily application of these two testing in laboratories was also understood through questionnaire surveys.In the first part of this study,nine encephalitis-related RNA viruses detection reference materials were established and external quality assessment was conducted.Viral encephalitis is a disease with high fatality rate.More than 100 viruses have been found to cause central nervous system infection.Except for some common encephalitis viruses,Chinese laboratories do not carry out routine testing for other encephalitis-related viruses that are less prevalent in the population.However,with the increase of population flow and entry and exit activities,the probability of these viruses spreading to China has been increased,so we need to be vigilant at all times.We selected 9 encephalitis-related RNA viruses that are less prevalent in the country but may pose a serious threat to public health,which were Yellow fever virus(YFV),West Nile virus(WNV)and Tick borne encephalitis virus(TBEV),Saint Louis encephalitis virus(SLEV),Venezuelan equine encephalitis virus(VEEV),Eastern equine encephalitis virus(EEEV),Western equine encephalitis virus(WEEV),Hendra virus(HeV)and Nipah virus(NiV).Real-time reverse transcription-polymerase chain reaction(rRT-PCR)is a common method for RNA nucleic acid detection.To date,some rRT-PCR kits for the nine viruses have been submitted for registration by several manufacturers in China.There are many steps in rRT-PCR,and factors such as sample preservation,RNA extraction,selection of commercial test kit,and test instrument platform will have a great impact on test results.Therefore,it is very important and necessary to develop reliable reference materials for EQA,to confirm whether the laboratories have accurate and reliable detection capability,and to promote the improvement of laboratory quality management.In this study,using molecular cloning,prokaryotic expression and protein purification techniques,based on the principle of specific binding of phage MS2 capsid protein to RNA,the RNA of 9 viruses were packaged into the capsid protein of MS2-VLP,and 9 recombinant MS2 VLPs were successfully prepared.The number of RNA copies in the recombinant MS2 VLP was quantified by droplet digital PCR(ddPCR)and then diluted to 102-105 copies/mL using simulated cerebrospinal fluid to form EQA disks of 26 samples.Finally,the samples were made into freeze-dried powder by vacuum lyophilization technology and distributed to laboratories all over the country to evaluate the nucleic acid detection capability of each laboratory,focusing on the detection and analytical performance of the laboratory,including analytical sensitivity and analytical specificity.A total of 31 laboratories participated in this EQA.96.3%(26/27),94.1%(15/16),100.0%(8/8),92.9%(13/14),100%(13/13),100.0%(5/5),82.4%(14/17),100%(8/8)and 100.0%(8/8)of laboratories completely correctly detected YFV,WNV,EEEV,WEEV,VEEV,SLEV,NiV,HeV and TBEV,respectively.Most(15/17,88.2%)of the reagents’sensitivity were greater than 90%,and the overall false positive rate and false negative rate were 0.3%and 0.7%.For EEEV and SLEV,the cycle threshold(Ct)of primer probe sets in different detection target areas was significantly different.In this study,9 viral encephalitis-related RNA viruses detection reference materials were successfully prepared.Accurate and reliable viral load values were obtained by digital PCR absolute quantification.And it has the advantages of safe and reliable without biological infection risk and mass production.In addition,this study is the first to carry out the EQA for the above viruses in China,and evaluate the viral nucleic acid detection capacity of laboratories and manufacturers,which is of guiding significance for ensuring public health safety and improving the virus detection capacity of laboratories i.The second part is to study the reference materials and external quality assessment of non-invasive prenatal testing for fetal chromosomal microdeletion/microduplication syndrome.Microdeletion/microduplication syndrome(MMS)caused by copy number variation(CNV)is one of the genetic factors leading to birth defects in newborns.Clinically common chromosome microdeletion/microduplication syndromes include 22q11 Deletion Syndrome((22q11DS),1p36 Deletion Syndrome(1p36DS),Cri du Chat Syndrome(CdCS)and Angelman Syndrome/Prader-Willi Syndrome(AS/PWS),etc.,which are often difficult to detect in early pregnancy due to the small number of missing fragments.At present,due to the improvement of library construction technology and the reduction of sequencing cost,the detection range of NIPT is gradually expanded,and the detection of MMS or CNV is gradually becoming possible.However,at present,none of NIPT product has been approved by the FDA.There are many challenges in the application of NIPT after expanding the detection range.Firstly,compared with the numerical variation,the fragments of microdeletion and microduplication are smaller,which requires higher resolution.Secondly,the detection of low fetal fraction(FF)is a major challenge,which is prone to false negative results.At the same time,there are many NIPT detection platforms and the detection process is complex,and many factors may affect the detection.In addition,the incidence,pathogenicity,clinical sensitivity,clinical specificity,positive predictive values(PPVs),and negative predictive values(NPVs)and other parameters for CNVs are not fully recognized by mostly laboratories,and reliable advice in genetic counseling cannot provided,which will eventually cause anxiety,increased economic burden and waste of medical resources.Therefore,the preparation of reference materials similar to clinical samples for EQA is the most critical premise to promote the standardization of NIPT and understand the current status of NIPT testing in Chinese laboratories.In this study,based on the enzymatic digestion technology which simulating the process of cfDNA,we prepared NIPT reference materials for five common microdeletion syndromes.The simulated fetal cfDNA was added to the plasma of clinical pregnant women,and a total of 12 EQA samples with FF range from 5%to 15%were prepared.After validation by NGS,a nationwide EQA campaign was carried out.The 12 samples together with the case information and questionnaires were distributed to the laboratories nationwide.The detection capacity and application of NIPT in microdeletion and microduplication were evaluated according to the detection rate,false negative rate and false positive rate.A total of 69 laboratories participated in this EQA.91.30%(63/69)of laboratories correctly identified all samples,while 7.25%(5/69)of laboratories reported false negative results and 2.90%(2/69)of laboratories reported false positive results.The detection rates of 22q11DS,CdCS,1p36DS and AS/PWS samples were 97.46%,98.55%,100%and 100%,respectively.With the increase of CNV fragment size,FF value and sequencing depth,the detection rate of each sample increased.For CNV sizes,the results showed that the false negative results were all from the samples with CNV size ≤7Mb.For sequencing depth,83.3%(10/12)of false negative results came from laboratories with unique reads less than 3.5M.In addition,only 7 laboratories reported FF values,and none of them reported sensitivity,specificity,positive predictive value and negative predictive value.In summary,this is the first preparation of non-invasive prenatal testing reference materials for fetal chromosomal microdeletion and microduplication syndrome,which can simulate the real clinical samples to the greatest degree.The reference materials have stable production process,can be mass produced,and are widely applicable to existing detection methods.At the same time,this study is the first EQA for five common fetal chromosomal microdeletion and microduplication syndromes(22q11DS,1p36DS,CdCS,AS and PWS)in China,evaluating the detection capabilities of different laboratories and different platforms,and determining the impact of FF,CNV size and sequencing depth on the detection of laboratories.It is helpful for laboratory to find related problems and provides a theoretical basis for clinical application of NIPT for microdeletion and microduplication.