A Novel Dual-specificity Probe Real-time Reverse Transcriptase-PCR (DSPrtRT-PCR) Using Dual-specific Armored RNA As Internal Control For HIV-1 Detection And Non-infectious RNAse-resistant Reference Material And Control Preparation For HIV-1 RNA Detection

A Novel Dual-specificity Probe Real-time Reverse Transcriptase-PCR(DSPrtRT-PCR) Using Dual-specific Armored RNA as Internal Control forHIV-1 Detection and Non-infectious RNAse-resistant Reference Material andControl Preparation for HIV-1 RNA DetectionABSTRACTObjectiveTo establish a novel dual-specificity probe real-time reversetranscriptase-PCR(DSPrtRT-PCR)using dual-specific armored RNA as internalcontrol for HIV-1 detection, in order to increase the positive ratio. Todevelop the virus-like particles (VLPs) Reference Material which containthe HIV-1 recombinant RNA (reRNA) and was ribonuclease resistant andnon-infectious. The Non-infectious RNAse-resistant Reference Materialpreparation for HIV-1 RNA detection could act as positive controls andexternal quality assessment (EQA) sample for clinical lab.Methods1. A Novel Dual-specificity Probe Real-time Reverse rranscriptase-PCR(DSPrtRT-PCR) Using Dual-specific Armored RNA as Internal Control forH[V-1 Detection(1) The alignment of consensus HTV-1 sequences of the Los AlamosNational Laboratory was downloaded from http://hiv-web, lanl. gov. HIV-1sequences were compared by using DANMAN and Bioedit software. Then theconsensus sequences of HIV-1 genome were determined. Oligonucleotides weredesigned with Primer Express software (Applied Biosystems). The degree ofnucleotide sequence homology of all oligonucleotides to sequences otherthan the HIV-1 sequence was checked by the EMBL, GenBank databases and theBLAST algorithm. The real-time reverse transcriptase-PCR for HIV-1detection was developed.(2) The selected primers and probes were screened by the detectionof patient sample and the virus-like particles (VLPs) Reference Material.The dual-specificity probe real-time reverse transcriptase-PCR (DSPrtRT-PCR) by two different probes and two pairs of primers wasdeveloped for HIV-1 detection. Based on the two different probes and twopairs of primers, the recombinant plasmid pET-MS2-Internal was constructedand identified. Then the virus-like particles (Armored RNA) as internalcontrol was expressed and purified. The concentration of Armored RNA usedas an internal control for detection of patient plasma by real-time RT-PCRwith TaqMan probes was determined. The optimization of multiplex RT-PCRwas carried out by the different additive and enzyme mixture adjustment.(3) The dual-specificity probe real-time reverse transcriptase-PCR(DSPrtRT-PCR) by two different probes and two pairs of primers and usingdual-specific armored RNA as internal false-negative control for HIV-1detection was developed. The sensitivity and specificity of the DSPrtRT-PCRdetection was tested. Furhtmore, the sensitivity, clinical fitness anddifferent group HIV-1 detection were compared the real-time PCR withmono-specificity probe with the DSPrtRT-PCR.2. The recombinant protein COAT expression and purification,anti-COATprotein polyclonal antibody preparation and recombinant protein COATinteraction with the RNA molecule transcribed in vitro(1) the recombinant plasmids pET₄₂-COAT,pcDNA3.1-COAT andpGEX-4t-1-COAT were constructed. The recombinant protein COAT wasexpressed in E. Coli. and purified by Ni⁺ chromatography. The purifiedprotein was dialyzed by urea-fold dilution.(2) The anti-COAT antibody was prepared by immunization withrecombinant protein COAT and plasmid pcDNA3. 1-COAT. The No. land No. 3 rabbitwere immunized with recombinant protein COAT; the No. 2 and No. 4 rabbit wereimmunized with plasmid pcDNA3. 1-COAT.(3) The TMK buffer was prepared. The interaction between MS2 phagecoat protein and RNA containing packaging site transcribed in vitro by RNAelectrophoretic mobility shift assay.3. The non-infectious RNAse-resistant Reference Material containing a part of the sequence of HIV-1 RNA preparation and the EQA for HIV-1 RNAtest in clinical labs(1) An expression system to produce the non-infectiousRNAse-resistant Reference Material containing a part of the sequence ofHIV-1 RNA was constructed, which were ribonuclease resistant. Thenon-infectious RNAse-resistant Reference Material was expressed andpurified in large scale.(2) The homogeneity of the Non-infectious RNAse-resistant ReferenceMaterial were tested. The stability of Reference Material was evaluatedat room temperature (20-25℃),37℃, 2-8℃and at-20℃. The stability ofReference Material was also evaluated when freeze-thaw cycles was repeated5 times. The concentration of Reference Material was quantified by HIV-1RNAdetection kit provided by PiJi Corporation and COBAS Ampicor HIV-1 1.5Monitor kit.(3) The EQA involved in HIV-1 RNA test was developed, which includedtwenty samples and report forms for each clinical lab.Results1. A Novel Dual-specificity Probe Real-time Reverse Transcriptase-PCR(DSPrtRT-PCR) Using Dual-specific Armored RNA as Internal Control forHIV-1RNA DetectionBy the alignment of consensus HIV-1 sequences 10 pairs of primers andprobes were designed. And two different probes and two pairs of primerswere screened by the detection of patient sample and the virus-likeparticles (VLPs) Reference Material for HIV-1 detection and the DSPrtRT-PCRwas developed, 1, O00copies/ml of armored RNA as internal control was usedas an internal control with the DSPrtRT-PCR detection. The limit ofdetection was about 125IU/ml HIV-1 RNA; the specificity of DSPrtRT-PCR was100% for the HIV-negative plasma; Compared with the real-time PCR withmono-specificity probe, all of the HIV-1 group M and group O could bedetected by DSPrtRT-PCR; all of the nine HIV-1 prevalent subtypes B, C. CRF01-AE,CRF07-BC, CRF08-BC could be detected by DSPrtRT-PCR; thesensitivity of detection was no significant difference betweenmono-specificity probe and DSPrtRT-PCR assays. When the DSPrtRT-PCRdetection was used 50 samples were positive and the other 10 samples wereconfirmed negative by COBAS AmpliScreen on 60 plasma samples, but when thereal-time PCR with mono-specificity probe was used, only 39 samples werepositive.2. The recombinant protein COAT expression and purification,anti-COATprotein polyclonal antibody preparation and recombinant protein COATinteraction with the RNA molecule transcribed in vitroThe expression and purification of recombinant protein COAT weresuccessfully acquired. By immunization with the purified recombinantprotein COAT and recombinant plasmid pcDNA3.1-COAT in rabbit, the antiserumwas harvested. Furthermore, the antiserum tater of rabbit immunization withthe purified recombinant protein COAT was 1:1280000, that of immunizationwith plasmid pcDNA3.1-COAT was only 1:8000. The mobility of RNA containingpackaging site changed.3. The non-infectious RNise-resistant Reference Material containinga part of the sequence of HIV-1 RNA preparation and the EQA for HIV-1 RNAtest in clinical labsNo significant change was observed when the Non-infectiousRNise-resistant Reference Material for HIV-1 RNA detection was preservedat different temperature. The Non-infectious RNise-resistant ReferenceMaterial for HIV-1 RNA detection was stable at room temperature (20-25℃)for 3 weeks, at 37℃for 10 days, at 2-8℃and at-20℃for more than 4 weeks.No significant change was observed when freeze-thaw cycles was repeated5 times. The homogeneity of the Non-infectious RNAse-resistant ReferenceMaterial was fine, which applicability to the clinical lab and EQA wasevaluated well. However, the main problem in pathogen nucleic acids wasthe detection of low concentration sample by the EQA. In order to assure the quality of clinical pathogen nucleic acids detection, the qualitycontrol and the EQA in clinical lab should be strengthened.ConclusionsThe DSPrtRT-PCR proves to be a more efficient and effective viral assaycharacterized with high sensitivity, specificity and feasibility comparedwith the conventional PCR with mono-specificity probe. As such, it can bewidely used for blood donor screening and individual qualitative detectionof HIV-1 RNA.The stability, homogeneity and applicability of the Non-infectiousRNAse-resistant Reference Material was verified well, it would bebeneficial to the quality control and EQA involved in HIV-1 RNA test.【Key words】HIV-1, dual-specificity probe, real-time reverse transcriptase-PCR,reference material...