Rapid Detection Of Four Respiroviruses By Multiplex Real-time Fluorescence Quantitative RT-PCR

BackgroundAcute respiratory tract infections (ARTIs) is a severe population health problemall over the world, it is also the second leading death cause among children under fiveyears’ old. The main pathogens of ARTIs include viruses, bacteria, mycoplasma,chlamydia and so on, among which viruses turns out to be of great significance.According to statistics, about90%of cases are caused by respiroviruses.Establishing a rapid and accurate differential diagnosis technology can be veryhelpful in real-time monitoring of respiroviruses outbreak, which provides a scientificevidence for the adjusting and improving of the corresponding disease control andprevention measures to stop the disease from spreading around during the earlyepidemic, and which provides a scientific evidence for the ecaluation about thecontrol and prevention measures. Establishing a rapid and accurate differentialdiagnosis technology can also perform a role in the early stage clinical etiologydiagnosis and the therapeutic regimen, it can reduce the morbidity and the mortality,it can reduce the disease burden and the economic burden for the nation and the patients themselves, it clearly has important social significance. Establishing a rapidand accurate differential diagnosis technology can also take effect in diseaseprevention and control system, immigration inspection and quarantine system andclinical examination system, it has great practical value and popularizing significance.The differential diagnosis technology of respiroviruses can be divided into twocategories: the traditional methods and the molecular biological methods. Thetraditional methods has too many flaws and deficiencies to meet the requirements ofquick and accurate respiroviruses screening. With the rapid development of molecularbiology, more and more new methods are used in the respiroviruses differentialdiagnosis field recently. Due to the high sensitivity and specificity, the achieve ofmultiple reactions, the automation, non-polluting, timeliness and accuracy, real-timefluorescence quantitative PCR and real-time fluorescence quantitative RT-PCR havebecome the fastest-developing and the most mature techonology in the field ofmolecular diagnostics. There are many kinds of respiroviruses, including DNA virusesand RNA viruses, in order to detect two types of viruses simultaneously, Real-timefluorescence quantitative RT-PCR can be used in the differential diagnosis ofrespiroviruses.Multiplex real-time fluorescence quantitative RT-PCR technology is atechnology developed on the basis of real-time fluorescence quantitative RT-PCRtechnology, by selecting multiple fluorescence channels and adding more pairs ofprimers and more probes (labeled with different fluorophores), this technologyenables the detection of a variety of nucleic acid templates, which means it canaccomplish the molecular differential diagnosis of a variety of respiroviruses.Currently, due to the quantitative limitation of fluorescent RT-PCR equipments’detection channels, multiplex fluorescence detection system can only achieve thesimultaneous detection of four or five target sequences, in addition to relying onscientific and technological progress to increase the quantitative limitation ofdetection channels, other methods must be came up with to increase the detectionnumber of target sequences. On this basis, multicolor recombination probe coding(MCPC) technology has been proposed. Meanwhile, homo-tag assisted non-dimer (HAND) system can greatly reduce the formation of primer dimers by adding samelabel to the5’-ends of upstream primers and downstream primers respectively toreduce the interference of primer-dimers in multiplex real-time fluorescencequantitative RT-PCR system.Experimental studies abroad shows that it is possible to detect three kinds offluorophors simultaneously using the current widely used multi-channel real-timequantitative RT-PCR instruments. Along with MCPC techonology and HAND system,it is possible to achieve the simultaneous detection of seven kinds of nucleic acidtemplates using the current widely used multi-channel real-time RT-PCR instrument,and it can break through the detection channel limitation of real-time fluorescencequantitative RT-PCR instruments, decrease the formation of primer dimmers andreduce the effect primer dimers has on the fluorescence RT-PCR reactions.Based on multiplex real-time fluorescence quantitative RT-PCR technology,MCPC technology and HAND system, this study is performed to establish a HANDmultiplex real-time fluorescence quantitative RT-PCR system for the detection ofhuman adenovirus (HADV), human bocavirus (HBoV), human metapneumovirus(HMPV) and human respiratory syncytial virus (HRSV), and to establish thedifferential diagnosis standard of the real-time fluorescence quantitative RT-PCRsystem for these4respiroviruses. This study provides a possible approach for therapid and accurate multiplex differential diagnosis technology of all kinds ofrespiroviruses.Methods1. The design and synthesis of specific binding primers, tailed primers and probes:download certain numbers of gene sequences of HADV, HBoV, HMPV, HRSVfrom Genebank, look for the highly conserved region of the gene sequences todesign specific binding primers and TaqMan probes. Tailed primers are designedby adding an universal primer sequence to the5’-ends of specific binding primers’ sequences. Synthesize specific binding primers, tailed primers andprobes.2. The positive clones of target sequences and the in vitro transcribed RNAs: usethe coverage gene fragment of the primer pairs as target sequences. ChooseTakara Biotechnology (Dalian) as authorized company to accomplish the positiveclones of target sequences and tha in vitro transcribed RNAs.3. The preparation of clinical specimens templates: use the clinical swab samples ofHADV, HBoV, HMPV, HRSV and other kinds of respiroviruses that causessimilar clinical symptoms as samples, use Roche’s High Pure Viral Nucleic AcidKit to extract viral nucleic acid templates.4. The establish of real-time fluorescence quantitative RT-PCR system: determinethe initial reaction system components and reaction conditions, accomplishreal-time fluorescence quantitative RT-PCR experiments. Set temperaturegradient to determine the annealing and extending temperature of the reactions.Use106-101copies/μl in vitro transcribed RNA to explore the sensitivity of thereal-time fluorescence quantitative RT-PCR systems. Complete the standardcurve to calculate the amplification efficiency of real-time fluorescencequantitative RT-PCR reaction systems. Set3parallel sample holes, calculate themean values and standard deviations of the3CT values, obtain the coefficient ofvariations of the3CT values, obtain the reproducibility of the reaction systems.Extract nuclein acid templates from the clinical samples to test the specificity ofthe reaction systems.5. The establish of multiplex real-time fluorescence quantitative RT-PCR system:determine the initial reaction system components and reaction conditions,accomplish real-time fluorescence quantitative RT-PCR experiments. Use106-101copies/μl in vitro transcribed RNA to explore the sensitivity of themultiplex real-time fluorescence quantitative RT-PCR systems. Complete thestandard curve to calculate the amplification efficiency of multiplex real-timefluorescence quantitative RT-PCR reaction systems. Set3parallel sample holes,calculate the mean values and standard deviations of the3CT values, obtain the coefficient of variations of the3CT values, obtain the reproducibility of thereaction systems. Extract nuclein acid templates from the clinical samples to testthe specificity of the reaction systems.6. The establish of HAND multiplex real-time fluorescence quantitative RT-PCRsystem: explore the concentration of tailed primes and probes of these4kinds ofrespiroviruses. Use106-101copies/μl in vitro transcribed RNA to explore thesensitivity of the HAND multiplex real-time fluorescence quantitative RT-PCRsystems. Complete the standard curve to calculate the amplification efficiency ofHAND multiplex real-time fluorescence quantitative RT-PCR reaction systems.Set3parallel sample holes, calculate the mean values and standard deviations ofthe3CT values, obtain the coefficient of variations of the3CT values, obtain thereproducibility of the reaction systems. Extract nuclein acid templates from theclinical samples to test the specificity of the reaction systems.Results1. The establish and examine of real-time fluorescence quantitative RT-PCRsystems: the real-time fluorescence quantitative RT-PCR systems of HADV,HBoV, HMPV, HRSV were successfully established. When the annealing andextending temperature reached60℃, the reaction systems were still able to detectthe in vitro transcribed RNA accurately. The sensitivity of detecting HADV,HBoV, HMPV and HRSV were10copies/μl identically, the coefficients ofdetermination were: HADV0.9998, HBoV0.9981, HMPV0.9981, HRSV0.9947and0.9965． The amplification efficiencies were: HADV105.08%, HBoV107.81%, HMPV102.08%, HRSV106.38%and107.95%, respectively. Thevalues of variation coefficients of real-time fluorescent quantitative RT-PCR allremain in a low level in the detection of6different concentrations of the vitrotranscribed RNA, and the specificity of this assay reached100%in clinicalsamples testing. 2. The establish and examine of multiplex real-time fluorescence quantitativeRT-PCR systems: the multiplex real-time fluorescence quantitative RT-PCRsystems of HADV, HBoV, HMPV, HRSV were successfully established. Thesensitivity of detecting HADV, HBoV, HMPV and HRSV were10copies/μlidentically, the coefficients of determination were: HADV0.9985, HBoV0.9998,HMPV0.9965, HRSV0.9966and0.9954．The amplification efficiencies were:HADV103.63%, HBoV102.65%, HMPV100.37%, HRSV105.54%and104.07%, respectively. The values of variation coefficients of multiplex real-timefluorescent quantitative RT-PCR all remain in a low level in the detection of6different concentrations of the vitro transcribed RNA, and the specificity of thisassay reached100%in clinical samples testing.3. The establish and examine of HAND multiplex real-time fluorescencequantitative RT-PCR systems: the optimum volumes of tailed primers were:HADV0.15μl, HBoV0.10μl, HMPV0.10μl, HRSV0.10μl, the optimumvolumes of probes were: HADV0.30μl, HBoV0.25μl, HMPV0.30μl, HRSV0.15μl for each probe and0.30μl in total. The sensitivity of detecting HADV,HBoV, HMPV and HRSV were102copies/μl identically, the coefficients ofdetermination were: HADV0.9977, HBoV0.9969, HMPV0.9985, HRSV0.9969and0.9979．The amplification efficiencies were: HADV99.58%, HBoV96.45%,HMPV98.64%, HRSV93.32%and94.62%, respectively. The values of variationcoefficients of HAND multiplex real-time fluorescent quantitative RT-PCR allremain in a low level in the detection of5different concentrations of the vitrotranscribed RNA(106-102copies/μl), and the specificity of this assay reached100%in clinical samples testing.Conclusions1. Successfully established the real-time fluorescence quantitative RT-PCR systemsof HADV, HBoV, HMPV, HRSV, the sensitivity of the systems have reached10 copies/μl identically, and the systems have achieved high amplification efficiency,reproducibility and specificity.2. Successfully established the multiplex real-time fluorescence quantitativeRT-PCR systems of HADV, HBoV, HMPV, HRSV, the sensitivity of the systemshave reached10copies/μl identically, and the systems have achieved highamplification efficiency, reproducibility and specificity.3. Successfully established the HAND multiplex real-time fluorescence quantitativeRT-PCR systems of HADV, HBoV, HMPV, HRSV, the sensitivity of the systemshave reached102copies/μl identically, which reduced1order of magnitude, butthe systems still have achieved comparatively high sensitivity, and the systemshave achieved high amplification efficiency, reproducibility and specificity. Thesystems need to be optimized in follow-up study, hoping to improve thesensitivity and amplification efficiency of the systems.4. This system based on multiplex real-time fluorescence quantitative RT-PCRtechnology can be used as a rapid and sensitive method in the detection of HADV,HBoV, HMPV and HRSV. It requires only2hours from the preparation oftemplates to the obtain of results. It provides a possible approach for the rapidand accurate multiplex differential diagnosis of all kinds of respiroviruses.