WO2019001187A1 - 一种快速区分五种小鼠呼吸道病原的多重液相基因芯片检测引物、试剂盒及方法 - Google Patents

一种快速区分五种小鼠呼吸道病原的多重液相基因芯片检测引物、试剂盒及方法 Download PDF

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WO2019001187A1
WO2019001187A1 PCT/CN2018/088302 CN2018088302W WO2019001187A1 WO 2019001187 A1 WO2019001187 A1 WO 2019001187A1 CN 2018088302 W CN2018088302 W CN 2018088302W WO 2019001187 A1 WO2019001187 A1 WO 2019001187A1
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primer
detection
liquid phase
tag sequence
kit
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王静
郭鹏举
黄韧
张钰
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广东省实验动物监测所
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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    • C12Q2600/00—Oligonucleotides characterized by their use
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Definitions

  • the invention belongs to the field of pathogenic microorganism detection of experimental animals, and particularly relates to a rapid distinction between five mouse respiratory pathogens - mouse pneumonia virus (PVM), hantavirus (HV), Sendai virus (SV), lymphocytic choroid plexus membrane. Multi-liquid phase microarray detection primers, kits and methods for inflammatory virus (LCMV) and Mycoplasma pneumoniae (MP).
  • PVM mouse respiratory pathogens - mouse pneumonia virus
  • HV hantavirus
  • SV Sendai virus
  • LCMV inflammatory virus
  • MP Mycoplasma pneumoniae
  • Microbial quality testing of experimental animals is an important indicator for evaluating the quality of experimental animals.
  • the national standard for the prevention of microorganisms in mice without specific pathogens includes 16 kinds of bacteria and 11 kinds of viruses.
  • the microbial quality test of experimental animals is to measure these pathogens carried by experimental animals by using certain detection means. It mainly includes two aspects of pathogenic detection and serological detection.
  • Conventional methods for pathogen detection, including bacterial culture, virus isolation and culture, and other methods for direct detection of pathogens are the gold standard for detection methods, but they are time consuming and labor intensive.
  • Serological diagnosis is the detection of pathogen infections that have occurred in animal populations by detecting serum antibody levels in animals.
  • the detection method has a long detection period, low sensitivity, low specificity, inability to detect dead materials, and failure to diagnose early viral infections.
  • China's experimental animal microbial detection is mainly through bacterial culture, biochemical identification, serum antibodies ELSIA, IFA, IEC and other conventional techniques, one for each pathogen detection, a SPF animal quality testing requires 27 projects The single test is very time consuming and labor intensive, and the demand for sample size is also large.
  • Real-time quantitative PCR realizes the quantification of the target molecule by directly detecting the change of the fluorescent signal during the PCR reaction. It does not require electrophoresis detection, and the whole process is completely closed-tube operation, the probability of contamination is reduced, and the false positive problem easily caused by conventional PCR is avoided. .
  • real-time fluorescent PCR has advantages in sensitivity, specificity and speed.
  • real-time fluorescent PCR technology is limited by the type of fluorescence and the instrument itself. At most, only 5 targets can be detected, and the success of the experiment is extremely difficult. Big.
  • the object of the present invention is to provide a multi-liquid phase gene chip detection primer, kit and method for rapidly distinguishing five mouse respiratory pathogens.
  • a multi-liquid phase microarray detection primer for rapidly distinguishing five mouse respiratory pathogens the nucleotide sequence of which is as follows:
  • A1 5'-AGATCACAGAGCCCGTCAAAAT-3' (SEQ ID NO: 1);
  • A2 5'-GCATATAACATCCAATACGAGTTTGAA-3' (SEQ ID NO: 2);
  • D1 5'-GAGTCCAGAAGCTTTCTGATGTCAT-3' (SEQ ID NO: 7);
  • D2 5'-CAAGTATTCACACGGCATGGAT-3' (SEQ ID NO: 8);
  • E1 5'-AGCGTTTGCTTCACTTTGAA-3' (SEQ ID NO: 9);
  • E2 5'-GGGCATTTCCTCCCTAAGCT-3' (SEQ ID NO: 10).
  • primers A1, B1, C1, D1 and E1 are also linked by a spacer sequence to a tag sequence.
  • the tag sequences at the 5' end of primers A1, B1, C1, D1 and E1 are respectively:
  • the tag sequence of primer A1 is: 5'-TACTTCTTTACTACAATTTACAAC-3' (SEQ ID NO: 11);
  • the tag sequence of primer B1 is: 5'-TACTTAAACATACAAACTTACTCA-3' (SEQ ID NO: 12);
  • the tag sequence of primer C1 is: 5'-CTAAACATACAAATACACATTTCA-3' (SEQ ID NO: 13);
  • the tag sequence of primer D1 is: 5'-CACTACACATTTATCATAACAAAT-3' (SEQ ID NO: 14);
  • the tag sequence of primer E1 was: 5'-ACTTATTTCTTCACTACTATATCA-3' (SEQ ID NO: 15).
  • primers A2, B2, C2, D2 and E2 is labeled with a biotin label.
  • a multiple liquid phase gene chip detection kit for rapidly distinguishing five mouse respiratory pathogens which comprises the primer according to any one of the above.
  • the kit further comprises a streptavidin-phycoerythrin complex and five fluorescent coded microspheres encoding different fluorescent colors.
  • the fluorescently encoded microspheres are coated with an anti-tag sequence that is complementary to the tag sequence in the primer.
  • a multi-liquid phase gene chip detection method for rapidly distinguishing five kinds of mouse respiratory pathogens comprising the following steps:
  • the hybridization product is analyzed to determine the type of pathogen in the test sample.
  • the hybridization reaction system and procedure are:
  • the total volume is 100 ⁇ L
  • the reaction was carried out at 45 ° C for 30 min.
  • the method of the invention can simultaneously detect mouse pneumonia virus, hantavirus, Sendai virus, lymphocytic choriomeningitis virus, and M. pneumoniae
  • the target amplified fragment is obtained by PCR, and then the amplification product and the fluorescent code are micro-coded.
  • the ball is hybridized with streptavidin-phycoerythrin (SA-PE), and then different types of pathogens are resolved when the MFI value is read by the detector.
  • SA-PE streptavidin-phycoerythrin
  • the method of the present invention combines a multi-liquid phase gene chip (MFIA) and a TAG technology, and the TAG technology uses Luminex's proprietary universal tag to perform nucleic acid experiment optimization and product development through unique complementary pairing of the tag sequence and the anti-tag sequence. And molecular diagnostic tests.
  • TAG technology guarantees the same renaturation temperature and hybridization efficiency, and effectively avoids cross-hybridization between microspheres labeled with different detectors.
  • the method of the invention can simultaneously detect mouse pneumonia virus, Hantavirus, Sendai virus, lymphocytic choriomeningitis virus, and M. pneumoniae, and has strong specificity, high sensitivity and good repeatability. Compared with the conventional detection method, the method of the invention realizes simultaneous detection of a plurality of different target molecules in the same sample, the sample amount is small, the operation is simple and fast, and the detection cost can be greatly reduced.
  • the primer of the invention has good amplifying and specificity for mouse pneumonia virus, hantavirus, Sendai virus, lymphocytic choriomeningitis virus, and M. pneumoniae, in addition to being combined with the above pathogens, It does not bind to other common mouse respiratory viruses and bacterial nucleic acids, and has high specificity and high accuracy.
  • the size of the amplified target fragments can be distinguished, and can be confirmed by conventional electrophoretic detection while being resolved by the fluorescent reporter signal.
  • Figure 1 shows the specific amplification electrophoresis pattern of MP single-weight primers for 5 pathogen cDNA templates; Lanes 1 to 5 are PCR amplification results of PVM, SV, LCMV, HV, MP cDNA template MP single-strand primers, lanes 6, 7
  • M is a 500 bp DNAmark.
  • Lane 5 is positive for M.P., the amplification product is about 300 bp in size, and other non-specific templates are negative.
  • Figure 2 is a detailed amplification electrophoresis map of five pathogen cDNA templates of PVM and SV single primers; lanes 1 to 7 are PCR amplification results of PVM single primers, and the templates are PVM, SV, LCMV, HV, MPcDNA.
  • blank control, blank control, corresponding lane 1 is PVM positive, the amplification product size is about 146 bp, other non-specific templates are negative; lanes 8 to 12 are SV single-primer PCR amplification electrophoresis results, the template is PVM, SV, LCMV, HV, MPcDNA, blank control, blank control; Lane 8 is SV positive, the amplification product size is about 196 bp, other non-specific templates are negative, M is 500 bp DNAmark.
  • Figure 3 is a detailed amplification electrophoresis pattern of LCMV and HV single-weight primers for five pathogen cDNA templates; lanes 1 to 7 are PCR amplification results of LCMV single-primer primers, and the templates are sequentially PVM, SV, LCMV, HV, MPcDNA.
  • blank control, blank control, corresponding lane 3 is LCMV positive, the amplification product size is about 162 bp, other non-specific templates are negative; lanes 8 to 12 are HV single-primer PCR amplification electrophoresis results, the template is PVM, SV, LCMV, HV, MPcDNA, blank control, blank control; Lane 10 is HV positive, the amplification product size is about 131 bp, other non-specific templates are negative, M is 500 bp DNAmark.
  • Figure 4 is a multiplex PCR electrophoresis map of five pathogens in the respiratory tract of mice; M is 500 bp Marker, 5 kinds of mouse respiratory pathogen primers are mixed, and 5 single PCR amplification is performed on single template.
  • Lane 1 is PVM template
  • lane 2 For the SV template
  • lane 3 is the LCM template
  • lane 4 is the HV template
  • lane 5 is the MP template
  • lane 6 is the negative template control.
  • each positive template well could specifically amplify a single target band, and the target bands were 146 bp, 196 bp, 162 bp, 131 bp, and 300 bp in sequence, and there was no cross-reactivity between the five pairs of primers, and the specificity was good.
  • Fig. 5 is a multiplex PCR electrophoresis pattern of five kinds of pathogen artificial mixed mixed infection templates in mouse respiratory tract; 5 PCR reaction system for 1-5 heavy cDNA positive template multiplex PCR electrophoresis results, lane 1 is SV single weight template, target band size 196 bp Lane 2 is the mixed template detection of SV and PVM; the target band is PVM146bp and SV 196bp; lanes 3 and 4 are mixed detection of 3 templates, the target bands are PVM146bp, SV196bp, LCM162bp; lane 5 is quadruple template.
  • the target bands were PVM146bp, SV196bp, LCM162bp, HV131bp; Lane 6 was mixed detection of five templates, the target bands were PVM146bp, SV196bp, LCM162bp, HV131bp, MP300bp.
  • Figure 6 shows the results of multiplex fluorescence detection of five pathogens in the respiratory tract of mice; five pathogen-specific primers bind to the magnetic bead capture sequence through the taq tag to excite the labeled fluorescent signal; the MFI value is read by the lumines200 instrument, and each specificity is visible.
  • the template can be specifically amplified corresponding to the respective template, while the template-free product has no excitation fluorescence signal.
  • Figure 7 is a multiplex fluorescence-specific detection map of five pathogens in the respiratory tract of mice; PVM, SV, HV, MP, LCMV, EDIM, TMEV, MVN, MHV, Reo3, ECT, MAD, Poly, MVM as specific Sex template, specific analysis of the xTAG detection method using the established method.
  • the results showed that the PVM, SV, HV, LCMV, MP and 5-fold positive cDNA mixed templates detected positive fluorescent signals corresponding to the labeled magnetic beads.
  • the detection methods were 8 for Reo3, TMEV, MHV, MNV, MAD, MVM, Poly and Ect.
  • Non-specific template controls have no cross-reactivity and are more specific.
  • Figure 8 is a PCR analysis of the multi-liquid phase microarray detection sensitivity of five pathogens in the respiratory tract of mice; lanes 1-11 are mixed plasmids of 5 heavy plasmids, corresponding copy number is 10 9 -10 -2 copies, corresponding to lane 8 (10 2 The copies) are the detection limits of the method.
  • Figure 9 shows the results of multiplex fluorescence detection of multi-liquid phase microarray detection of five pathogens in the respiratory tract of mice.
  • the experimental results show that the sensitivity detection limits of PVM, SV, LCMV and MP are 10 2 copies/ ⁇ l, and the detection MFI value is about 250-1389, the sensitivity detection limit of HV is 10 3 copies/ ⁇ l, the detection MFI value is about 962, and the MFI detection result is consistent with the gel electrophoresis result.
  • Figure 10 shows the results of 5-fold Xtaq fluorescence detection in SV artificially infected mice.
  • the results show that the tissue samples of artificially infected mice can be detected on the 3rd to 7th day after infection by liquid phase gene chip fluorescence detection of five pathogens.
  • SV was detected positive, the toxic dose reached a peak, and began to decline on the 10th day.
  • the tissues with higher toxic content have lung, heart, spleen and kidney.
  • the four pathogens PVM, LCMV, M.P., and HV were negative.
  • Figure 11 shows the results of the HV project for the xtaq detection of the HV clinical sample; the MFI value of the 5-fold liquid phase gene chip of J18-4 and APR-32 is less than 500, and the result is negative; the others are positive.
  • Figure 12 is a comparison of the results of the M.P. project of the 5th taq detection of the clinical sample with the QPCR M.P. test results. The results show that the lower the Ct value of the M.P. detection, the higher the corresponding MFI detection value, and the results of the two methods are consistent.
  • Figure 13 shows the results of 5-fold fluorescence taq detection in lung tissue of clinical mice.
  • Example 1 A multi-liquid phase microarray detection primer for rapidly distinguishing five mouse respiratory pathogens
  • primer pairs A1 and A2, B1 and B2, C1 and C2, D1 and D2, E1 and E2 were found to simultaneously detect mouse pneumovirus (PVM) and Hantavirus (HV).
  • PVM mouse pneumovirus
  • HV Hantavirus
  • Sendai virus (SV) Sendai virus
  • LCMV lymphocytic choriomeningitis virus
  • MP Mycoplasma pneumoniae
  • the nucleotide sequence of the primer pair used to detect mouse pneumovirus (PVM) is as follows:
  • A1 5'-AGATCACAGAGCCCGTCAAAAT-3' (SEQ ID NO: 1);
  • A2 5'-GCATATAACATCCAATACGAGTTTGAA-3' (SEQ ID NO: 2);
  • the nucleotide sequence of the primer pair used to detect Hantavirus (HV) is as follows:
  • the nucleotide sequence of the primer pair used to detect Sendai virus (SV) is as follows:
  • LCMV lymphocytic choriomeningitis virus
  • D1 5'-GAGTCCAGAAGCTTTCTGATGTCAT-3' (SEQ ID NO: 7);
  • D2 5'-CAAGTATTCACACGGCATGGAT-3' (SEQ ID NO: 8);
  • the nucleotide (M.P.) sequence of the M. pneumoniae used for detection is as follows:
  • E1 5'-AGCGTTTGCTTCACTTTGAA-3' (SEQ ID NO: 9);
  • E2 5'-GGGCATTTCCTCCCTAAGCT-3' (SEQ ID NO: 10).
  • a tag sequence is attached to the 5' end of the primers A1, B1, C1, D1 and E1 via a spacer arm, which is an intermediate modification group added between the two nucleic acid sequences, which provides necessary for oligonucleotide labeling. Intervals to reduce the interaction between the labeling group and the oligonucleotide. Spacer 18 is used in the present invention.
  • the tag sequence of primer A1 is: 5'-TACTTCTTTACTACAATTTACAAC-3' (SEQ ID NO: 11);
  • the tag sequence of primer B1 is: 5'-TACTTAAACATACAAACTTACTCA-3' (SEQ ID NO: 12);
  • the tag sequence of primer C1 is: 5'-CTAAACATACAAATACACATTTCA-3' (SEQ ID NO: 13);
  • the tag sequence of primer D1 is: 5'-CACTACACATTTATCATAACAAAT-3' (SEQ ID NO: 14);
  • the tag sequence of primer E1 was: 5'-ACTTATTTCTTCACTACTATATCA-3' (SEQ ID NO: 15).
  • Biotin labeling was added to the 5' end of primers A2, B2, C2, D2 and E2.
  • Example 2 A multi-liquid phase microarray detection kit for rapidly distinguishing five mouse respiratory pathogens
  • the test kit includes the following components:
  • the liquid phase gene chip kit of the present invention comprises the above detection system, magnetic beads (ie, fluorescent coded microspheres), streptavidin-phycoerythrin complex, reagent and the like, and the solid phase chip is distinguished from the solid phase chip.
  • the detection probe is a taq sequence designed on a primer, and hybridization is combined with the complementary sequence of the magnetic beads, and the detection process is performed by reading the fluorescent signal on the magnetic beads on the hybridization.
  • Example 3 Establishment of a multi-liquid phase microarray detection method for five mouse respiratory pathogens
  • RNA/DNA was extracted from five pathogenic nucleic acids in the respiratory tract of mice, and RT reverse transcription was performed to prepare a positive cDNA specific template.
  • RNA reverse transcription reaction system is as follows:
  • the single PCR reaction system is as follows:
  • RNA/DNA was extracted from five pathogenic nucleic acids in the respiratory tract of mice, and RT reverse transcription was performed to prepare a positive cDNA specific template.
  • Preparation of the upstream primer mixture mixing A1, B1, C1, D1 and E1 in a 1:1 ratio;
  • the multiplex PCR amplification reaction procedure was: pre-denaturation at 94 ° C for 5 min; denaturation at 94 ° C for 30 s, annealing at 58 ° C for 60 s, extension at 72 ° C for 30 s for 35 cycles; and 72 ° C for 7 min.
  • Single-plex template 5 re-primer multiplex PCR reaction multiplex PCR product was analyzed by 4% agarose gel electrophoresis, and the electrophoresis pattern is shown in Fig. 4.
  • M is a 500 bp Marker
  • 5 mouse respiratory pathogen primers are mixed, and 5 single PCR amplification is performed on the single template.
  • Lane 1 is a PVM template
  • Lane 2 is an SV template
  • Lane 3 is an LCM template
  • Lane 4 is a LCM template.
  • HV template lane 5 is the MP template
  • lane 6 is the negative template control.
  • the results of Fig. 4 show that each positive template well can specifically amplify a single target band.
  • the size of the target band is 146 bp, 196 bp, 162 bp, 131 bp, and 300 bp.
  • the five pairs of primers have no cross-reactivity and specificity.
  • the 5-fold PCR reaction system can specifically amplify the target fragment for 1-5 heavy cDNA positive template, and the target band size is single-plate template SV196bp, 2-fold template PVM146bp, SV 196bp; 3-fold template PVM146bp, SV196bp, LCM162bp; quadruple template PVM146bp, SV196bp, LCM162bp, HV131bp; five-fold template PVM146bp, SV196bp, LCM162bp, HV131bp, MP300bp.
  • microspheres with specific anti-tag sequences are coated separately (the coating process can be processed by themselves, or the luminex company can be purchased directly), and the anti-tag sequence can be corresponding to PVM, HV, LCMV.
  • the tag sequences on the five pathogen primers of SV, and MP are complementary paired.
  • Five kinds of microspheres were purchased from luminex, and the corresponding PVM, HV, LCMV, SV, and MP corresponding fluorescent coded microspheres were MTAG-A015, MTAG-A065, MTAG-A042, MTAG-A062 and MTAG-A034.
  • the five pathogen-specific primers bind to the magnetic bead capture sequence through the taq tag to excite the labeled fluorescent signal; the MFI value is read by the lumines200 instrument and the results are shown in FIG. It can be seen in Figure 6 that each specific template can specifically amplify the template, while the template-free product (ie, the negative sample ntc) has no excitation fluorescence signal, and its MFI is below the cutoff value.
  • the detection method can obtain the detection results of five pathogens by one PCR, and the specificity is good.
  • the PCR product hybridizes with the fluorescent coded microsphere working solution, streptavidin phycoerythrin (SA-PE) working solution, and includes the following steps:
  • SA-PE working solution preparation 1 mg/ml SA-PE was diluted to 10 ⁇ g/ ⁇ l with 1 ⁇ Tm Hybrdization Buffer.
  • cutoff value 30 SPF mouse lung tissue samples were selected (each sample was repeated in parallel 3 times), and the MFI values were read and the mean and standard deviation were calculated. It is set to the cutoff value by the MIF value of the average plus 3 times the standard deviation. The cutoff value obtained by this method is shown in Table 1.
  • the cutoff value of the corresponding PVM item is set to 200, M.P. is set to 200, LCMV is set to 250, HV is set to 500, and SV is set to 250.
  • M.P. is set to 200
  • LCMV is set to 250
  • HV is set to 500
  • SV is set to 250.
  • Example 4 Multi-liquid phase microarray detection method for five kinds of mouse respiratory pathogens
  • PVM, SV, HV, M.P., LCMV, EDIM, TMEV, MVN, MHV, Reo3, ECT, MAD, Poly, MVM were used as specific templates to analyze the established multi-liquid phase microarray detection method.
  • Each pathogenic nucleic acid was extracted with the Tiangen Nucleic Acid Automatic Extractor Virus DNA/RNA Co-extraction Kit, reverse transcription was performed using the Takara RT kit, and the viral cDNA was detected by Qiagen multiplex PCR kit.
  • the results are shown in Figure 7.
  • the PVM, SV, HV, LCMV, EDIM, MP, and 5-fold positive cDNA mixed templates correspond to labeled magnetic beads to detect positive fluorescent signals.
  • the detection methods are for Reo3, TMEV, MHV, MNV, MAD, MVM, Poly, and The eight non-specific template controls of Ect did not cross-react.
  • the specificity test confirmed that the detection method did not cross-react with other mouse pathogens.
  • the detection system has good specificity.
  • Example 5 Multi-liquid phase microarray detection method for five kinds of mouse respiratory pathogens
  • RNA/DNA of PVM, HV, LCMV, SV, and MP pathogens were extracted by Tiangen's automatic nucleic acid extractor, and the corresponding primers designed in Example 1 were used for RT-PCR amplification, respectively. It was detected by agarose gel electrophoresis and purified by gelatinization.
  • the purified cDNA was ligated into the pMD-20T vector using the TaKaRa kit, and the ligated product was transformed into DH5a competent cells, and the monoclonal antibody was selected for colony PCR identification, and the colonies identified as positive bacteria were subjected to plasmid extraction. Send sequencing.
  • the plasmid was extracted using a plasmid extraction kit, and the concentration and purity of the positive plasmid were determined by a micro ultraviolet spectrophotometer, and the copy number was calculated according to the following formula.
  • Copy number (copies/ ⁇ L) 6.022 ⁇ 10 23 (copies/mol) ⁇ DNA concentration (g / ⁇ L) / mass MW (g / mol).
  • MW number of DNA bases (bp) ⁇ 660 daltons / bp
  • number of DNA bases number of bases of the vector sequence + number of bases of the inserted sequence.
  • Each of the prepared plasmids was quantified. The quantified plasmids were each mixed in an appropriate amount so that the copy number of each plasmid of the mixed template was 10 10 copies/ul.
  • Multi-liquid phase microarray detection method Sensitivity test: Multiple plasmid templates were diluted by a 10-fold dilution method and diluted to 10 -2 copies/ ⁇ l. The 10 9 -10 -2 copies/ ⁇ l multiplex plasmid template was detected by the multi-liquid phase gene chip method established above. The results of the sensitivity test of the 5 heavy liquid gene microarray in mouse respiratory pathogen are shown in Fig. 8 and Fig. 9. In Fig. 8, lanes 1-11 are mixed plasmids of 5 heavy plasmids, and the corresponding copy number is 10 9 -10 -2 copies. Corresponding to lane 8 (10 2 copies) is the detection limit of the method.
  • Example 6 Detection of SV artificial infection samples
  • the experimental samples of artificially infected SV pathogens were detected by the established 5-fold mouse respiratory pathogen liquid phase gene chip detection method, and compared with the QPCR test results. The results are shown in Fig. 10.
  • the detection results of 96 artificially infected SV tissue samples were consistent with QPCR, and the tissue toxicity reached the peak on the 3rd to 7th day after infection, and began to decrease on the 10th day.
  • the tissues with higher toxic content had lung, heart, spleen and kidney, while the four pathogens PVM, LCMV, MP and HV were negative (Fig. 10).
  • the tissue samples of artificially infected PVM pathogens were detected by the established 5-fold mouse respiratory pathogen liquid phase gene chip detection method, and compared with the results of QPCR detection. After testing, the detection results of 72 artificially infected PVM tissue samples were consistent with QPCR, the positive rate of QPCR detection was 3/72, and the xtaq multiplex fluorescent PCR detection was also positive for 3 samples, and the other samples were tested. Negative, negative, positive coincidence rate of 100%.
  • a unit in Zhejiang provided 13 clinical HV suspicious samples.
  • the test results of this unit were 5 HV negative and 8 HV positive.
  • the multi-liquid phase microarray detection method of the present invention was used to detect the results.
  • the results shown in Fig. 11 showed that 2 HV were negative. 10 HV positive, positive coincidence rate of 7/8, negative compliance rate of 1/5.
  • Example 9 Detection of lung tissue samples from KM mice fed an open environment
  • the lung tissue samples of 51 KM mice raised in an open environment of a clinical unit were extracted, and the multi-liquid phase microarray detection method of the present invention was used for detection.
  • the detection results showed that 51 lung tissue samples were detected by 5 respiratory pathogens, and all SV were negative.
  • PVM was all negative
  • MP was positive
  • HV background was high
  • LCM test results were all negative
  • Figure 13 is the comparison between the test results and QPCR detection method, the results are the same.

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Abstract

本发明公开了一种快速区分5种小鼠呼吸道病原的多重液相基因芯片检测引物、试剂盒及方法。本发明通过PCR获得目标扩增片段，然后将扩增产物、荧光编码微球和链霉亲和素‑藻红蛋白进行杂交，通过检测仪读取MFI值，从而分辨不同类型的病毒。

Description

一种快速区分五种小鼠呼吸道病原的多重液相基因芯片检测引物、试剂盒及方法 技术领域
本发明属于实验动物的病原微生物检测领域，具体涉及一种快速区分5种小鼠呼吸道病原-小鼠肺炎病毒(PVM)、汉坦病毒(HV)、仙台病毒(SV)、淋巴细胞脉络丛脑膜炎病毒(LCMV)、肺支原体(M.P.)的多重液相基因芯片检测引物、试剂盒及方法。
背景技术
实验动物作为研究材料，其标准化程度至关重要，是研究数据和结论科学性、重复性的重要保障。实验动物微生物质量检测是评价实验动物质量的重要指标，我国国标规定无特定病原体的小鼠需排除的微生物包括16种细菌和11种病毒。实验动物微生物质量检测就是通过采用一定的检测手段对实验动物携带的这些病原进行测定。主要包括病原学检测和血清学检测两方面技术。病原学检测方面常规方法包括细菌培养、病毒分离培养等方法直接检测病原体，是检测方法的金标准，但比较费时费力。血清学诊断则是通过检测动物血清抗体水平，发现在动物群体中已经发生过的病原感染。该检测方法检测周期长、灵敏度不高、特异性不强、不能检测病死材料，而且无法对早期病毒感染作出诊断。目前，因条件所限，我国实验动物微生物检测主要还是通过细菌培养，生化鉴定、血清抗体ELSIA、IFA、IEC等常规技术，对每个病原逐一检测，一个SPF动物的质量检测需要进行27个项目的单项检测，非常耗时耗力，而且对样品量的需求也较大。
随着分子生物学的迅速发展，以PCR技术为基础的各种分子生物学诊断技术，成为生物医学领域内最有价值的研究手段和病毒学诊断的金标准，已经广泛的应用于临床标本的检测。多重PCR通过在同一个反应中同时完成多个基因的扩增成为临床与基础研究领域中一种简便快捷的筛选检测方法。多重PCR技术结合了病原分子检测高敏感性、高特异性和高通量的优势，是临床多种疾病混合感染诊断的理想技术，但也存在很多问题，如在结果判定方面，传统多重PCR检测，需要设计扩增不同大小PCR片段通过电泳区别，费时费力，反应产物容易产生污染而导致假阳性，而且多重反应中扩增片段大小差异过大，PCR效率不一，易造成结果存在偏差。荧光定量PCR则通过直接检测PCR反应过程中荧光信号的变化实现对目的分子的定量，不需要电泳检测，并且整个过程完全闭管式操作，污染机率降低，避免了常规PCR容易造成的假阳性问题。相对常规PCR，荧光定量PCR在敏感性、特异性与速度等 方面具有优势，但实时荧光PCR技术受到荧光种类及仪器自身的限制，最多只能对5个靶标进行检测，且实验成功的难度极大。
发明内容
本发明的目的在于提供一种快速区分五种小鼠呼吸道病原的多重液相基因芯片检测引物、试剂盒及方法。
本发明所采取的技术方案是：
一种快速区分5种小鼠呼吸道病原的多重液相基因芯片检测引物，该引物的核苷酸序列如下所示：
A1：5’-AGATCACAGAGCCCGTCAAAAT-3’(SEQ ID NO：1)；
A2：5’-GCATATAACATCCAATACGAGTTTGAA-3’(SEQ ID NO：2)；
B1：5’-GGACACAATCAATGGGGATACAAC-3’(SEQ ID NO：3)；
B2：5’-CCATATCATCCCCTAAGTGGAA-3’(SEQ ID NO：4)；
C1：5’-CCCAGCCATATACTCAGTCGTGC-3’(SEQ ID NO：5)；
C2：5’-TCCACAACTTTTGTGACAGGACAC-3’(SEQ ID NO：6)；
D1：5’-GAGTCCAGAAGCTTTCTGATGTCAT-3’(SEQ ID NO：7)；
D2：5’-CAAGTATTCACACGGCATGGAT-3’(SEQ ID NO：8)；
E1：5’-AGCGTTTGCTTCACTTTGAA-3’(SEQ ID NO：9)；
E2：5’-GGGCATTTCCTCCCTAAGCT-3’(SEQ ID NO：10)。
优选的，引物A1、B1、C1、D1和E1的5’端还通过间隔臂连接有tag序列。
优选的，引物A1、B1、C1、D1和E1的5’端的tag序列分别为：
引物A1的tag序列为：5’-TACTTCTTTACTACAATTTACAAC-3’(SEQ ID NO：11)；
引物B1的tag序列为：5’-TACTTAAACATACAAACTTACTCA-3’(SEQ ID NO：12)；
引物C1的tag序列为：5’-CTAAACATACAAATACACATTTCA-3’(SEQ ID NO：13)；
引物D1的tag序列为：5’-CACTACACATTTATCATAACAAAT-3’(SEQ ID NO：14)；
引物E1的tag序列为：5’-ACTTATTTCTTCACTACTATATCA-3’(SEQ ID NO：15)。
优选的，引物A2、B2、C2、D2和E2的5’端添加有生物素标记。
一种用于快速区分5种小鼠呼吸道病原的多重液相基因芯片检测试剂盒，该试剂盒中包含上述任一项所述的引物。
优选的，该试剂盒中还含有链霉亲和素-藻红蛋白复合物、5种编码不同荧光色的荧光编码微球。
优选的，荧光编码微球上包被有与引物中tag序列互补配对的anti-tag序列。
一种快速区分5种小鼠呼吸道病原的多重液相基因芯片检测方法，包括如下步骤：
1)从样品中提取病毒RNA/DNA，以RNA/DNA为模板，加入上述任一项所述的引物进行RT-PCR扩增；
2)将扩增产物、5种编码不同荧光色的荧光编码微球和链霉亲和素-藻红蛋白复合物进行杂交；
3)杂交结束后，对杂交产物进行分析，确定检测样本中的病原类型。
优选的，杂交的反应体系和程序为：
5种荧光编码微球20μL
链霉亲和素-藻红蛋白复合物75μL
扩增产物5μL
总体积100μL；
45℃反应30min。
本发明的有益效果是：
本发明方法可以同时对小鼠肺炎病毒、汉坦病毒、仙台病毒、淋巴细胞脉络丛脑膜炎病毒、鼠肺支原体进行检测时，通过PCR获得目标扩增片段，然后将扩增产物、荧光编码微球和链霉亲和素-藻红蛋白(SA-PE)进行杂交，然后通过检测仪读取MFI值时，分辨不同类型的病原。
本发明的方法将多重液相基因芯片(MFIA)和TAG技术结合起来，TAG技术使用Luminex专有的通用标签，通过标签序列与反标签序列的专一性互补配对，进行核酸实验优化，产品开发和分子诊断化验。TAG技术能保证相同的复性温度和杂交效率，且有效避免不同检测物标记的微球之间交叉杂交。
本发明方法，能够同时对小鼠肺炎病毒、汉坦病毒、仙台病毒、淋巴细胞脉络丛脑膜炎病毒、鼠肺支原体进行检测，而且特异性强，灵敏度高，重复性好。与传统检测方法相比，本发明方法实现了对同一样本中的多种不同目的分子同时进行检测，样本用量少，操作简单、快速，可大大降低检测成本。
本发明的引物，对小鼠肺炎病毒、汉坦病毒、仙台病毒、淋巴细胞脉络丛脑膜炎病毒、鼠肺支原体，均有很好的扩增性及特异性，除可以与上述病原结合外，不与其他常见的小鼠呼吸道病毒及细菌核酸结合，特异性强，准确性高。此外，所获扩增目的片段大小可以加以区分，在通过荧光报告信号分辨的同时还可通过传统的电泳检测加以确认。
附图说明
图1为M.P.单重引物对5种病原cDNA模板特异性扩增电泳图；泳道1至5依次为PVM、SV、LCMV、HV、M.P.cDNA模板M.P.单重引物PCR扩增结果，泳道6、7为阴性对照，M为500bpDNAmark。泳道5为M.P.阳性，扩增产物大小约为300bp，其他非特异模板均为阴性。
图2为PVM、SV单重引物对5种病原cDNA模板特异性扩增电泳图；泳道1至7为PVM单重引物PCR扩增电泳结果，模板依次为PVM、SV、LCMV、HV、M.P.cDNA、空白对照、空白对照，对应泳道1为PVM阳性，扩增产物大小约为146bp，其他非特异模板均为阴性；泳道8至12为SV单重引物PCR扩增电泳结果，模板依次为PVM、SV、LCMV、HV、M.P.cDNA、空白对照、空白对照；泳道8为SV阳性，扩增产物大小约为196bp，其他非特异模板均为阴性，M为500bpDNAmark。
图3为LCMV、HV单重引物对5种病原cDNA模板特异性扩增电泳图；泳道1至7为LCMV单重引物PCR扩增电泳结果，模板依次为PVM、SV、LCMV、HV、M.P.cDNA、空白对照、空白对照，对应泳道3为LCMV阳性，扩增产物大小约为162bp，其他非特异模板均为阴性；泳道8至12为HV单重引物PCR扩增电泳结果，模板依次为PVM、SV、LCMV、HV、M.P.cDNA、空白对照、空白对照；泳道10为HV阳性，扩增产物大小约为131bp，其他非特异模板均为阴性，M为500bpDNAmark。
图4为小鼠呼吸道5种病原单重模板多重PCR电泳图；M为500bp Marker，5种小鼠呼吸道病原引物混合后对单重模板进行5重PCR扩增，泳道1为PVM模板，泳道2为SV模板，泳道3为LCM模板，泳道4为HV模板，泳道5为M.P.模板，泳道6为阴性模板对照。结果可见各阳性模板孔均可特异扩增单一目的条带，目的条带大小依次为146bp、196bp、162bp、131bp、300bp，五对引物间无交叉反应性，特异性较好。
图5为小鼠呼吸道5种病原人工模拟混合感染模板多重PCR电泳图；5重PCR反应体系对1-5重cDNA阳性模板多重PCR电泳结果，泳道1为SV单重模板，目的条带大小196bp；泳道2为SV、PVM的2重模板混合检测；目的条带为PVM146bp、SV 196bp；泳道3、4为3重模板混合检测，目的条带为PVM146bp、SV196bp、LCM162bp；泳道5为四重模板混合检测，目的条带为PVM146bp、SV196bp、LCM162bp、HV131bp；泳道6为五重模板混合检测，目的条带为PVM146bp、SV196bp、LCM162bp、HV131bp、M.P.300bp。
图6为小鼠呼吸道5种病原液相基因芯片多重荧光检测结果；5种病原特异性引物通过taq标签与磁珠捕获序列结合，激发标记荧光信号；通过lumines200仪器读取MFI值，可见 各特异模板均能对应各自模板特异性扩增，而无模板产物无激发荧光信号。
图7为小鼠呼吸道5种病原液相基因芯片多重荧光特异性检测图；以PVM、SV、HV、M.P.、LCMV、EDIM、TMEV、MVN、MHV、Reo3、ECT、MAD、Poly、MVM作为特异性模板，用所建立的方法进行xTAG检测方法的特异性分析。结果可见，PVM、SV、HV、LCMV、M.P.及5重阳性cDNA混合模板对应标记磁珠检测到阳性荧光信号，该检测方法对Reo3、TMEV、MHV、MNV、MAD、MVM、Poly及Ect这8种非特异性模板对照无交叉反应，特异性较好。
图8为小鼠呼吸道5种病原的多重液相基因芯片检测灵敏度PCR电泳图；泳道1-11为5重质粒混合模板，对应拷贝数依次10 9-10 -2copies，对应泳道8(10 2copies)为该方法的检测极限。
图9为小鼠呼吸道5种病原的多重液相基因芯片检测灵敏度多重荧光检测结果；实验结果表明，PVM、SV、LCMV、M.P.的灵敏度检出限为10 2copies/μl，检测MFI值约为250-1389，HV的灵敏度检出限为10 3copies/μl，检测MFI值约为962，MFI检测结果与凝胶电泳结果一致。
图10为SV人工感染实验小鼠组织5重Xtaq荧光检测结果；结果可见，通过5种病原的液相基因芯片荧光检测，人工感染小鼠组织样本在感染后第3-7天各组织均能检测到SV阳性，含毒量达到高峰，第10天开始下降。含毒量较高组织有肺、心、脾、肾。而PVM、LCMV、M.P.、HV这四种病原为阴性。
图11为HV临床样本xtaq检测HV项目结果；J18-4、APR-32的5重液相基因芯片检测MFI值小于500，判定结果为阴性；其余为阳性。
图12为临床样品5重taq检测M.P.项目结果与QPCR M.P.检测结果比较；结果可见，M.P.检测Ct值越低，对应的MFI检测值越高，二种方法检测结果一致。
图13为临床小鼠肺组织5重荧光taq检测结果。
具体实施方式
下面结合实施例对本发明作进一步说明，但并不局限于此。
实施例1：一种快速区分5种小鼠呼吸道病原的多重液相基因芯片检测引物
经过对所设计的大量引物进行筛选后，发现引物对A1和A2、B1和B2、C1和C2、D1和D2、E1和E2对同时快速检测小鼠肺炎病毒(PVM)、汉坦病毒(HV)、仙台病毒(SV)、淋巴细胞脉络丛脑膜炎病毒(LCMV)、肺支原体(M.P.)的效果最好，所获扩增目的片段大小可以加以区分，在通过荧光报告信号分辨的同时还可通过传统的电泳检测加以确认。其核苷酸序 列如下所示：
用于检测小鼠肺炎病毒(PVM)的引物对的核苷酸序列如下所示：
A1：5’-AGATCACAGAGCCCGTCAAAAT-3’(SEQ ID NO：1)；
A2：5’-GCATATAACATCCAATACGAGTTTGAA-3’(SEQ ID NO：2)；
用于检测汉坦病毒(HV)的引物对的核苷酸序列如下所示：
B1：5’-GGACACAATCAATGGGGATACAAC-3’(SEQ ID NO：3)；
B2：5’-CCATATCATCCCCTAAGTGGAA-3’(SEQ ID NO：4)；
用于检测仙台病毒(SV)的引物对的核苷酸序列如下所示：
C1：5’-CCCAGCCATATACTCAGTCGTGC-3’(SEQ ID NO：5)；
C2：5’-TCCACAACTTTTGTGACAGGACAC-3’(SEQ ID NO：6)；
用于检测淋巴细胞脉络丛脑膜炎病毒(LCMV)的核苷酸序列如下所示：
D1：5’-GAGTCCAGAAGCTTTCTGATGTCAT-3’(SEQ ID NO：7)；
D2：5’-CAAGTATTCACACGGCATGGAT-3’(SEQ ID NO：8)；
用于检测的鼠肺支原体的核苷酸(M.P.)序列如下所示：
E1：5’-AGCGTTTGCTTCACTTTGAA-3’(SEQ ID NO：9)；
E2：5’-GGGCATTTCCTCCCTAAGCT-3’(SEQ ID NO：10)。
在引物A1、B1、C1、D1和E1的5’端通过间隔臂连接有tag序列，间隔臂是加在两段核酸序列间的一个中间修饰基团，可为寡核苷酸标记提供必要的间隔，以减少标记基团与寡核苷酸间的相互作用。本发明中用的是spacer 18。
引物A1、B1、C1、D1和E1的5’端的tag序列分别为：
引物A1的tag序列为：5’-TACTTCTTTACTACAATTTACAAC-3’(SEQ ID NO：11)；
引物B1的tag序列为：5’-TACTTAAACATACAAACTTACTCA-3’(SEQ ID NO：12)；
引物C1的tag序列为：5’-CTAAACATACAAATACACATTTCA-3’(SEQ ID NO：13)；
引物D1的tag序列为：5’-CACTACACATTTATCATAACAAAT-3’(SEQ ID NO：14)；
引物E1的tag序列为：5’-ACTTATTTCTTCACTACTATATCA-3’(SEQ ID NO：15)。
引物A2、B2、C2、D2和E2的5’端添加生物素标记。
实施例2：一种快速区分5种小鼠呼吸道病原的多重液相基因芯片检测试剂盒
检测试剂盒包括以下组分：
(1)实施例1所设计的的多重液相基因芯片检测引物；
(2)5种编码不同荧光色的包含有anti-tag序列的荧光编码微球，所述anti-tag序列能相 应地与多重液相基因芯片检测引物中的tag序列互补配对；五种微球均购自luminex公司，具体的PVM、HV、LCMV、SV、和M.P.分别对应的荧光编码微球号为MTAG-A015、MTAG-A065、MTAG-A042、MTAG-A062和MTAG-A034；
(3)链霉亲和素-藻红蛋白复合物；
(4)PCR扩增反应体系的其他试剂。
本发明的液相基因芯片试剂盒包括上述引物、磁珠(即荧光编码微球)、链霉亲和素-藻红蛋白复合物、试剂等整个检测体系，与固相芯片相区别的是，检测探针是在设计在引物上的taq序列，通过与磁珠的互补序列杂交结合，检测过程是通过读取杂交上的磁珠上的荧光信号进行判读。
实施例3：5种小鼠呼吸道病原的多重液相基因芯片检测方法的建立
1、单重引物RT-PCR扩增特异性验证
(1)阳性模板制备：将小鼠呼吸道五种病原核酸提取RNA/DNA，进行RT反转录，制备阳性cDNA特异性模板。
RNA反转录反应体系如下：
Figure PCTCN2018088302-appb-000001
(2)单重PCR特异性验证：分别以单重引物A1A2、B1B2、C1C2、D1D2、E1E2对PVM、SV、LCMV、HV、M.P.这5种病原cDNA进行PCR扩增，验证单重引物的特异性。
单重PCR反应体系如下：
Figure PCTCN2018088302-appb-000002
Figure PCTCN2018088302-appb-000003
结果见图1、图2、图3。图1、图2、图3结果表明，各特异性引物均能较好地扩增出特异病原核酸，而与其他4种病原无交叉反应，引物特异性较好。
2、5重引物的RT-PCR扩增
(1)单重阳性模板制备：将小鼠呼吸道五种病原核酸提取RNA/DNA，进行RT反转录，制备阳性cDNA特异性模板。
(2)多重cDNA模板制备：分别将阳性cDNA模板等量混合，依次混合2重模板PVM、SV；3重模板PVM、SV、LCM；四重模板PVM、SV、LCM、HV；五重模板PVM、SV、LCM、HV、M.P.；制备人工混合感染模板与多重PCR反应体系进行反应。
(3)五重混合引物制备：
上游引物混合液的制备：将A1、B1、C1、D1和E1以1:1比例进行混合；
下游引物混合液的制备：将A2、B2、C2、D2和E2以1:1比例进行混合。
用混合的5重引物对PVM、HV、LCMV、SV、和M.P.阳性cDNA单重模板和1-5重多重模板进行五重PCR扩增。
(4)多重PCR反应体系如下：
Figure PCTCN2018088302-appb-000004
(5)多重PCR扩增的反应程序为：94℃预变性5min；94℃变性30s，58℃退火60s，72℃延伸30s，共35个循环；72℃终延伸7min。
(6)结果：
单重模板5重引物多重PCR反应：多重PCR产物进行4％琼脂糖凝胶电泳分析，电泳图如图4所示。图4中，M为500bp Marker，5种小鼠呼吸道病原引物混合后对单重模板进行5重PCR扩增，泳道1为PVM模板，泳道2为SV模板，泳道3为LCM模板，泳道4为HV模板，泳道5为M.P.模板，泳道6为阴性模板对照。图4结果可见各阳性模板孔均可特异扩 增单一目的条带，目的条带大小依次为146bp、196bp、162bp、131bp、300bp，五对引物间无交叉反应性，特异性较好。
多重模板多重PCR反应：依次以单重模板、2重模板、3重模板、4重模板、5重模板与5重PCR反应体系进行反应，结果如图5所示。图5中，5重PCR反应体系对1-5重cDNA阳性模板均可特异性扩增目的片段，目的条带大小依次为单重模板SV196bp、2重模板PVM146bp、SV 196bp；3重模板PVM146bp、SV196bp、LCM162bp；四重模板PVM146bp、SV196bp、LCM162bp、HV131bp；五重模板PVM146bp、SV196bp、LCM162bp、HV131bp、M.P.300bp。
3、液相基因芯片杂交检测：
分别包被有特异的anti-tag序列的6种微球(包被过程可自行处理，或直接购买luminex公司包被好的磁珠)，其中anti-tag序列能相应地与PVM、HV、LCMV、SV、和M.P.五种病原引物上的tag序列互补配对。五种微球均购自luminex公司，具体的PVM、HV、LCMV、SV、和M.P.分别对应的荧光编码微球号为MTAG-A015、MTAG-A065、MTAG-A042、MTAG-A062和MTAG-A034。5种病原特异性引物通过taq标签与磁珠捕获序列结合，激发标记荧光信号；通过lumines200仪器读取MFI值，结果见图6。图6中可见各特异模板均能对应各自模板特异性扩增，而无模板产物(即阴性样本ntc)无激发荧光信号，其MFI为cutoff值以下。本检测方法可通过一次PCR得到5种病原的检测结果，特异性较好。
PCR产物与荧光编码微球工作液、链霉亲和素藻红蛋白(SA-PE)工作液杂交，包括以下步骤：
1)荧光编码微球工作液的制备：将荧光编码微球用1×Tm Hybrdization Buffer稀释至1ul含每种荧光编码微球约125个。
2)SA-PE工作液制备：将1mg/ml SA-PE用1×Tm Hybrdization Buffer稀释到10μg/μl。
充分重悬荧光编码微球工作液，每个样品孔和背景孔加入微球工作液20μl，样品孔中加入5μl PCR产物，背景孔中加入5μl PCR blank产物，再加入75μl的SA-PE工作液，充分混匀，于金属加热器中45℃孵育30min。
3)依照检测仪Luminex 200仪器的说明将杂交后的50μl上述反应液进行检测，结果如图6所示，用检测仪Luminex 200仪器读取MFI值，可通过荧光编码分辩不同类型的病原。
4、液相基因芯片杂交检测结果判断标准(注：该判断标准仅供参考，亦可对结果判断标准进行调整)：
最低检测阈值(cutoff值)的确定：选取30份SPF小鼠肺组织样品(每个样品平行重复 3次)，分别读取MFI值并计算其平均值和标准差。以平均值加3倍标准差的MIF值设定其为cutoff值。通过该方法获得cutoff值如表1所示。
表1、5种小鼠呼吸道病原的多重液相基因芯片检测方法的cutoff值
  PVM M.P. LCMV SV HV
平均值 55.64 64.24 53.16 79.21 111.70
SD 46.90 42.47 51.61 54.25 126.72
平均值+3SD 196.33 191.64 208.00 241.96 491.87
Cutoff 200 200 250 250 500
待测样本的结果判定：
1)本发明中对应PVM项目cutoff值定为200、M.P.定为200、LCMV定为250、HV定为500、SV定为250。当待测样本的各病原对应荧光微球MFI值＞cutoff值时，判断为阳性样本。
2)待测样本的MFI值≤cutoff时，判断为阴性样本，需要进行重复实验或采取其他检测方法进一步验证。
实施例4：5种小鼠呼吸道病原的多重液相基因芯片检测方法特异性实验
以PVM、SV、HV、M.P.、LCMV、EDIM、TMEV、MVN、MHV、Reo3、ECT、MAD、Poly、MVM作为特异性模板，对所建立的多重液相基因芯片检测方法进行特异性分析。用天根核酸自动提取仪病毒DNA/RNA共提取试剂盒提取各病原核酸，takara RT试剂盒进行反转录，Qiagen多重PCR试剂盒对病毒cDNA进行检测。结果见图7。图7中，PVM、SV、HV、LCMV、EDIM、M.P.及5重阳性cDNA混合模板对应标记磁珠检测到阳性荧光信号，该检测方法对Reo3、TMEV、MHV、MNV、MAD、MVM、Poly及Ect这8种非特异性模板对照无交叉反应。通过特异性试验验证了该检测方法与其他小鼠病原无交叉反应。说明检测体系特异性良好。
实施例5：5种小鼠呼吸道病原的多重液相基因芯片检测方法灵敏性实验
1、5种小鼠呼吸道病原质粒的构建
用天根的核酸自动抽取仪分别提取PVM、HV、LCMV、SV、和M.P.病原的RNA/DNA，分别以实施例1所设计的对应的引物，进行RT-PCR扩增，将扩增产物分别进行琼脂糖凝胶电泳检测并切胶纯化。用TaKaRa公司的试剂盒将纯化后的cDNA连接至pMD-20T载体中，将连接产物转化至DH5a感受态细胞，挑选单克隆，进行菌落PCR鉴定，将鉴定为阳性菌的菌落进行质粒抽提，送测序。
2、多重反应模板的制备：
用质粒提取试剂盒提取质粒，微量紫外分光光度计测定阳性质粒浓度与纯度，根据下面 的公式计算拷贝数。拷贝数(copies/μL)＝6.022×10 23(copies/mol)×DNA浓度(g/μL)/质量MW(g/mol)。其中，MW＝DNA碱基数(bp)×660daltons/bp，DNA碱基数＝载体序列碱基数+插入序列碱基数。对制备的各质粒进行定量。定量好的质粒各取适量混合，使混合模板的各质粒的拷贝数均为10 10copies/ul。
3、多重液相基因芯片检测方法敏感性试验：采用10倍稀释法对多重质粒模板进行倍比稀释，稀释至10 -2copies/μl。用上述建立的多重液相基因芯片方法对10 9-10 -2copies/μl多重质粒模板进行检测。小鼠呼吸道病原5重液相基因芯片检测灵敏性实验结果如图8、图9所示，图8中，泳道1-11为5重质粒混合模板，对应拷贝数依次10 9-10 -2copies，对应泳道8(10 2copies)为该方法的检测极限。图9结果表明，PVM、SV、LCMV、M.P.的灵敏度检出限为10 2copies/μl，检测MFI值约为250-1389，HV的灵敏度检出限为10 3copies/μl，检测MFI值约为962，MFI检测结果与凝胶电泳结果一致。
实施例6：SV人工感染样品的检测
用所建立的5重小鼠呼吸道病原液相基因芯片检测方法对人工感染SV病原的实验鼠组织样品进行检测，并与QPCR检测结果进行比对，结果见图10。经检测，本发明方法对96份人工感染SV组织样品检测结果与QPCR一致，感染后第3-7天组织含毒量达到高峰，第10天开始下降。含毒量较高组织有肺、心、脾、肾，而PVM、LCMV、M.P.、HV这四种病原为阴性(图10)。对应QPCR检测灵敏性达到10 2(Ct 34)以上样品本方法检测均为阳性；而QPCR检测灵敏性达到10 2～10 1(Ct 34-35)的样本，本检测方法检测为阴性，与敏感性测试实验结果一致。
实施例7：PVM人工感染样品的检测
用所建立的5重小鼠呼吸道病原液相基因芯片检测方法对人工感染PVM病原的实验鼠组织样品进行检测，并与QPCR检测结果进行比对。经检测，本方法对72份人工感染PVM组织样品检测结果与QPCR一致，QPCR检测阳性率为3/72，xtaq多重荧光PCR检测对应该3份样本也为检测阳性，其余样品两种检测方法均为阴性，阴性、阳性符合率为100％。
实施例8：HV临床阳性可疑样品实验室间比对
浙江某单位提供13份临床HV可疑样本，该单位检测结果为5份HV阴性，8份HV阳性，使用本发明多重液相基因芯片检测方法检测，结果见图11显示结果为2份HV阴性，10份HV阳性，阳性符合率7/8，阴性符合率1/5。
使用本发明方法同时检测到12份支原体阳性，一份支原体阴性，检测结果与QPCR方法进行验证结果一致。结果见图12。
后期验证浙江检测结果有误，其改进检测方法后与本发明检测方法结果一致。
实施例9：临床某开放环境饲养KM小鼠肺组织样本的检测
对临床某单位开放环境下饲养的51份KM小鼠肺组织样本，提取核酸，使用本发明多重液相基因芯片检测方法进行检测，检测结果51份肺组织样本5种呼吸道病原检测，SV全部阴性，PVM全部阴性，M.P.一份阳性，HV背景偏高，但检测结果为阴性；LCM检测结果全部阴性；图13为本方法检测结果与QPCR检测方法进行比对，二者结果一致。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (9)

  1. 一种快速区分五种小鼠呼吸道病原的多重液相基因芯片检测引物，其特征在于：该引物的核苷酸序列如下所示：
    A1：5’-AGATCACAGAGCCCGTCAAAAT-3’；
    A2：5’-GCATATAACATCCAATACGAGTTTGAA-3’；
    B1：5’-GGACACAATCAATGGGGATACAAC-3’；
    B2：5’-CCATATCATCCCCTAAGTGGAA-3’；
    C1：5’-CCCAGCCATATACTCAGTCGTGC-3’；
    C2：5’-TCCACAACTTTTGTGACAGGACAC-3’；
    D1：5’-GAGTCCAGAAGCTTTCTGATGTCAT-3’；
    D2：5’-CAAGTATTCACACGGCATGGAT-3’；
    E1：5’-AGCGTTTGCTTCACTTTGAA-3’；
    E2：5’-GGGCATTTCCTCCCTAAGCT-3’。
  2. 根据权利要求1所述的引物，其特征在于：引物A1、B1、C1、D1和E1的5’端还通过间隔臂连接有tag序列。
  3. 根据权利要求1或2所述的引物，其特征在于：引物A1、B1、C1、D1和E1的5’端的tag序列分别为：
    引物A1的tag序列为：5’-TACTTCTTTACTACAATTTACAAC-3’；
    引物B1的tag序列为：5’-TACTTAAACATACAAACTTACTCA-3’；
    引物C1的tag序列为：5’-CTAAACATACAAATACACATTTCA-3’；
    引物D1的tag序列为：5’-CACTACACATTTATCATAACAAAT-3’；
    引物E1的tag序列为：5’-ACTTATTTCTTCACTACTATATCA-3’。
  4. 根据权利要求1所述的引物，其特征在于：引物A2、B2、C2、D2和E2的5’端添加有生物素标记。
  5. 一种用于快速区分五种小鼠呼吸道病原的多重液相基因芯片检测试剂盒，其特征在于，该试剂盒中包含权利要求1-4任一项所述的引物。
  6. 根据权利要求5所述的试剂盒，其特征在于：该试剂盒中还含有链霉亲和素-藻红蛋白复合物、5种编码不同荧光色的荧光编码微球。
  7. 根据权利要求5或6所述的试剂盒，其特征在于：荧光编码微球上包被有与引物中tag序列互补配对的anti-tag序列。
  8. 一种快速区分五种小鼠呼吸道病原的多重液相基因芯片检测方法，其特征在于，包括如 下步骤：
    从样品中提取病毒RNA/DNA，以RNA/DNA为模板，加入权利要求1-4任一项所述的引物进行RT-PCR扩增；
    将扩增产物、5种编码不同荧光色的荧光编码微球和链霉亲和素-藻红蛋白复合物进行杂交；
    杂交结束后，对杂交产物进行分析，确定检测样本中的病原类型。
  9. 根据权利要求8所述的方法，其特征在于：杂交的反应体系和程序为：
    5种荧光编码微球 20μL
    链霉亲和素-藻红蛋白复合物 75μL
    扩增产物 5μL
    总体积 100μL；
    45℃反应 30min。
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