WO2020224195A1 - Combinaison amorce-sonde pour la détection et la distinction d'espèces aspergillus, kit, procédé de détection et leur utilisation - Google Patents

Combinaison amorce-sonde pour la détection et la distinction d'espèces aspergillus, kit, procédé de détection et leur utilisation Download PDF

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WO2020224195A1
WO2020224195A1 PCT/CN2019/112918 CN2019112918W WO2020224195A1 WO 2020224195 A1 WO2020224195 A1 WO 2020224195A1 CN 2019112918 W CN2019112918 W CN 2019112918W WO 2020224195 A1 WO2020224195 A1 WO 2020224195A1
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aspergillus
detection
probe
seq
primer
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王志贤
阎香言
盛长忠
周泽奇
粟艳
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丹娜（天津）生物科技有限公司
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    • C12R2001/66—Aspergillus
    • C12R2001/685—Aspergillus niger

Definitions

  • the application belongs to the field of biotechnology, and relates to a primer probe combination, kit, detection method and application for the detection of Aspergillus species, and specifically to a primer for the detection of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Aspergillus terreus Probe combination, kit, detection method and application thereof.
  • Aspergillus is a conditional pathogen, which is widely present in the living environment. It can produce conidia into the body with breathing, and mainly infect the lungs, skin or mucous membranes. Common pathogens include Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus and so on.
  • the detection methods for Aspergillus mainly include: (1) Pathogen culture and identification. Take samples from the affected area for smear or culture. The smear shows hyphae or Aspergillus spores. The culture shows the growth of Aspergillus. Aspergillus is a common contaminating bacteria. Smears or cultures must be repeated, multiple positives and the same strain are of diagnostic value, and the culture cycle is long.
  • Pathological examination taking biopsy of damaged tissue or lymph node, and confirming the diagnosis based on fungal morphology, but this method is invasive examination, which is poorly accepted by patients.
  • Immunological examination galactomannan is a highly specific and conservative polysaccharide in the cell wall of Aspergillus.
  • the real-time fluorescent PCR method improves the detection specificity through the combination of primers and probes, and provides a basis for clinical diagnosis.
  • the existing method has a long detection cycle, which is not conducive to clinical diagnosis and medication.
  • CN108070675A discloses a primer probe combination and a fluorescent quantitative PCR kit for simultaneously detecting three Aspergillus species, belonging to the technical field of in vitro molecular detection of pathogenic microorganisms.
  • This application provides a primer-probe combination for simultaneous detection of three Aspergillus species based on a fluorescent PCR method, the Aspergillus including Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger.
  • this application can only detect any one or a combination of Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger in the sample, and cannot detect and determine the specific strain, and this method cannot diagnose the presence of Aspergillus terreus.
  • CN106755379A discloses a fluorescent quantitative PCR method based on dimer mutation primers to quantitatively detect 4 Aspergillus species and synchronously perform genotyping.
  • the detection method includes 1) extracting DNA from samples to be tested and 4 standard strains 2) Prepare positive plasmid standards; 3) Run fluorescent quantitative PCR; 4) Data analysis.
  • the application also provides a detection kit for simultaneous quantification and genotyping of Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, and Aspergillus terreus, which includes an upstream primer labeled with a fluorescent reporter group and a quenching group The complementary strand of the labeled upstream primer and the downstream primer.
  • the primers of this application are designed with fluorescent groups, which affect the amplification of fluorescence quantitative PCR; secondly, the melting curve temperature of the product is too high, the difference in annealing temperature between species of Aspergillus is too small, and the sensitivity and specificity of Aspergillus typing identification Not high; in the end, only common primers are used without probes, and the complementary primers are also designed with mutation sites, resulting in amplification of non-specific products, and the method has poor specificity.
  • CN101038254A discloses a kit that specifically amplifies the genes of the Aspergillus ITS I interval, and the product size is 79bp, which can quickly and accurately detect a variety of common pathogenic Aspergillus (such as Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus and Aspergillus nidulans).
  • Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus and Aspergillus nidulans However, the application was unable to detect interspecies differences of Aspergillus.
  • CN102321738A discloses a universal primer for fluorescent quantitative PCR detection of Aspergillus, a detection probe and a detection kit, which identify Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus and Aspergillus niger through a pair of universal primers and 4 specific probes. Although it can detect interspecies differences of Aspergillus, this method requires four fluorescence channels for analysis, which increases the experimental period and cost.
  • this application provides a primer probe combination, kit, detection method and application for the detection of Aspergillus species, one detection to determine whether it is Aspergillus infection, can be in the same fluorescence channel It also distinguishes Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Aspergillus terreus.
  • this application provides a primer probe combination for the detection of Aspergillus species, including specific primers, detection probes and complementary probes for Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Aspergillus terreus, respectively.
  • the probe and the detection probe are not completely complementary paired.
  • multiplex PCR is used to simultaneously amplify the specific target sequences of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, which are common in the invasive Aspergillus genus, and then use the specially designed Taqman probe melting curve analysis and identification to realize the For multiple detection of common invasive Aspergillus species, the primer probe combination of the present application has good specificity and high sensitivity, and realizes the interspecies detection of Aspergillus.
  • the detection probe and the complementary probe are not completely complementary paired, and the non-complementary base positions are not located within the 5 base sequence of the 5'end or 3'end of the detection primer, and the non-complementary positions cannot be continuous.
  • Tm ⁇ H * /( ⁇ S * +RlnC T ), where ⁇ H * and ⁇ S * are the standard enthalpy change and entropy change of the hybridization reaction, respectively, R is the gas constant 1.987 cal/kmol, and C T is the mole of DNA molecule
  • concentration when the DNA molecule is an asymmetric sequence, its molar concentration is taken as CT/4
  • CT/4 concentration
  • nucleotide sequence of the specific primer of Aspergillus fumigatus is shown in SEQ ID NO.1-2
  • nucleotide sequence of the specific primer of Aspergillus flavus is shown in SEQ ID NO.3-
  • SEQ ID NO.4 the nucleotide sequence of the specific primer of Aspergillus niger is shown in SEQ ID NO. 5-6
  • nucleotide sequence of the specific primer of Aspergillus terreus is shown in SEQ ID NO. 7-8 Shown.
  • primers and probes exist in the same reaction system to detect different target sequences. It is necessary to balance the stability, reaction conditions, and amplification efficiency among various sequences. As the number of detected bacteria increases, the combination of primers and probes The difficulty of design is significantly increased and is restricted by various conditions.
  • specific primers are designed through multiple sequence alignments against Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus, so that multiple primers can stably exist in the same reaction In the system, and the amplification efficiency is consistent, the detection specificity and sensitivity are improved, and the cross effect between primers is avoided.
  • nucleotide sequence of the detection probe of Aspergillus fumigatus is shown in SEQ ID NO. 9, and the nucleotide sequence of the detection probe of Aspergillus flavus is shown in SEQ ID NO. 10.
  • Aspergillus niger The nucleotide sequence of the detection probe is shown in SEQ ID NO.11, and the nucleotide sequence of the detection probe of Aspergillus terreus is shown in SEQ ID NO.12.
  • the range of comparison bacteria includes: the range of comparison bacteria includes Aspergillus fumigatus AJ853744.1, AB197939.1, LC228654.1, LC171332.1, LN832990.1, etc. 264 sequences; Aspergillus flavus AJ853764.1, LT594458.1, LC133097.1, LC133096.1, LN832989.1 and 351 sequences; Aspergillus niger AJ853742.1, AJ280006.1, LC133093.1, LN832991.1, LM653115.
  • the region of the four Aspergillus specific target sequences to be tested preferably 18S rRNA and 28S rRNA sequence regions, four Taqman probes and four complementary sequences are designed and prepared respectively, and a pair is designed for the periphery of the detection probe sequence.
  • Primer sequence including one upstream primer and one downstream primer. Use the designed primers to perform single-plex or multiple single-color or multiple-multicolor real-time fluorescence quantitative PCR amplification reactions on specific targets.
  • nucleotide sequence of the complementary probe of Aspergillus fumigatus is shown in SEQ ID NO. 13
  • nucleotide sequence of the complementary probe of Aspergillus flavus is shown in SEQ ID NO. 14.
  • Aspergillus niger The nucleotide sequence of the complementary probe is shown in SEQ ID NO.15
  • nucleotide sequence of the complementary probe of Aspergillus terreus is shown in SEQ ID NO.16.
  • a complementary probe is designed for the detection probes of four different Aspergillus species, and the complementary probe and the detection probe are not completely complementary paired to form a double-stranded probe, so that the double-stranded probe for different Aspergillus species
  • the probe has different melting properties, and four different Aspergillus species can be distinguished by its characteristic melting temperature (Tm).
  • the primer probe combination further includes an internal reference system.
  • the internal reference system includes internal reference primers and internal reference probes.
  • nucleotide sequence of the internal reference primer is shown in SEQ ID NO. 17-18.
  • nucleotide sequence of the internal reference probe is shown in SEQ ID NO.19.
  • the 3'end of the detection probe of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Aspergillus terreus is modified with a quenching group, and the 5'end is modified with a fluorescent group.
  • the fluorophore includes ALEX-350, Alexa Fluor 488, CY3, FAM, VIC, TET, CALGold540, JOE, HEX, CALFluorOrange560, TAMRA, CALFluorRed590, ROX, CALFluorRed610, Any one or a combination of at least two of TexasRed, CALFluorRed635, Quasar670, CY5, CY5.5, LC RED640 or Quasar705.
  • the 5'ends of the detection probes for different Aspergillus species are all modified with fluorescent groups, which can be the same fluorescent group or different fluorescent groups.
  • the primer-probe combination designed in this application can be detected and analyzed in the same fluorescence detection channel. According to the melting temperature of the double-stranded probe formed by the detection probe and the complementary probe, it can be performed after the real-time fluorescence quantitative PCR amplification reaction.
  • the quenching group modified at the 3'end of the detection probe includes any one or a combination of at least two of TAMRA, DABCYL, BHQ-1, BHQ-2, BHQ-3 or Eclipse.
  • the fluorophore and quenching group used can be the same or different, but different fluorophores require different fluorescence detection channels, and different fluorophores are selected
  • the excitation light and absorption light spectrum range need to avoid interference.
  • the probe-modified fluorophore and quencher group for detecting different Aspergillus species belong to the same group.
  • the combination of the fluorescent group and the quenching group can be, typically, but not limited to, the fluorescent group FAM and the quenching group TAMRA; the fluorescent group HEX quenching group BHQ-1; the fluorescent group ROX and the quenching group BHQ-2; the fluorescent group CY5 and the quenching group BHQ-3, preferably the fluorescent group FAM and the quenching group TAMRA.
  • the 3'end of the complementary probe is modified with a quenching group.
  • the quenching group modified at the 3'end of the complementary probe includes any one or a combination of at least two of TAMRA, DABCYL, BHQ-1, BHQ-2, BHQ-3 or Eclipse.
  • the quencher group of the complementary probe and the detection probe are the same.
  • the quenching group of the probe and the quenching group of the complementary sequence can also be different, but it needs to match the spectral band of the fluorescent group.
  • the 3'end of the complementary probe can be selectively modified with a quencher group.
  • the detection probe is complementary to the target sequence.
  • the 5'end is labeled with a fluorescent group
  • the 3'end is labeled with a quenching group.
  • the 3'end of the complementary probe can be designed to contain a quenching group or not designed to contain a quenching group.
  • the reason for this phenomenon is that the linear single-stranded double-labeled oligonucleotide in the solution is randomly coiled: its two ends occasionally approach each other, causing the energy of the fluorescent group to be absorbed by the quenching group.
  • the labeled hybrid forces the two ends of the probe to separate, disrupting the interaction between the two end labels, thereby causing a change in the fluorescence value.
  • the complementary probe is labeled with a quenching group at the 3'end, the hybridization between the probes reduces the fluorescence value, and when the temperature is increased to denature the duplex, the fluorescence value increases. This is because the formation of the probe hybrid brings the quenching group and the fluorescent group close together, effectively quenching the fluorescent signal.
  • the present application provides a kit for the detection of Aspergillus species, the kit comprising the primer probe combination as described in the first aspect.
  • the kit further includes negative control, positive quality control and auxiliary reagents.
  • the negative control is a plasmid containing an Arabidopsis DNA fragment, and the nucleotide sequence of the Arabidopsis DNA fragment is shown in SEQ ID NO.20.
  • the positive quality controls are plasmids containing Aspergillus fumigatus DNA fragments, plasmids containing Aspergillus flavus DNA fragments, plasmids containing Aspergillus niger DNA fragments, and plasmids containing Aspergillus terreus DNA fragments.
  • both negative control and positive quality control use plasmid pMD19, which is commonly used in the art. It should be noted that any vector that satisfies the construction of negative control and positive quality control plasmids can be used to replace pMD19, and no special limitation is required here.
  • nucleotide sequences of the Aspergillus fumigatus DNA fragment, Aspergillus flavus DNA fragment, Aspergillus niger DNA fragment and Aspergillus terreus DNA fragment are as SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO. 23 and SEQ ID NO.24 are shown.
  • the auxiliary reagents include Taq enzyme, dNTP, MgCl 2 , DMSO and buffer.
  • the buffer includes Tris-HCl and KCl.
  • the concentration of Tris-HCl in the buffer is 15-20 mM, for example, it can be 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM.
  • the concentration of the KCl in the buffer is 100-200mM, for example, it can be 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM or 200mM.
  • the present application provides a method for detecting Aspergillus species by using the kit as described in the second aspect, and the method includes the following steps:
  • the final concentration of the DNA sample in step (2) is 0.1pg-10ng, for example, it can be 0.1pg, 0.5pg, 1pg, 2pg, 3pg, 4pg, 5pg, 10pg, 20pg, 30pg, 40pg, 50pg, 60pg, 70pg, 80pg, 90pg, 100pg, 200pg, 300pg, 400pg, 500pg, 600pg, 700pg, 800pg, 900pg, 1ng, 2ng, 3ng, 4ng, 5ng, 6ng, 7ng, 8ng, 9ng or 10ng.
  • the final concentration of the Taq enzyme in step (2) is 0.5-5U, for example, it can be 0.5U, 0.8U, 1U, 1.2U, 1.5U, 1.8U, 2U, 2.5U, 3U, 3.5 U, 4U, 4.5U or 5U.
  • the final concentration of the dNTP in step (2) is 0.1-5M, for example, it can be 0.1M, 0.3M, 0.5M, 0.8M, 1M, 1.2M, 1.5M, 1.8M, 2M, 2.5 M, 2.8M, 3M, 3.5M, 4M, 4.5M, 4.8M or 5M.
  • the final concentration of the dNTP in step (2) is 0.1-5M, for example, it can be 0.1M, 0.3M, 0.5M, 0.8M, 1M, 1.2M, 1.5M, 1.8M, 2M, 2.5 M, 2.8M, 3M, 3.5M, 4M, 4.5M, 4.8M or 5M.
  • the volume percentage of DMSO in step (2) is 1-8%, for example, it can be 1%, 2%, 3%, 4%, 5%, 6%, 7% or 8%, preferably Is 5%.
  • the final concentration of the specific primers of the four Aspergillus species described in step (2) is 50-500nM, for example, it can be 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 120nM, 150nM, 180nM, 200nM , 250nM, 300nM, 350nM, 400nM, 450nM or 500nM.
  • the final concentration of the four Aspergillus detection probes in step (2) is 50-500nM, for example, it can be 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 120nM, 150nM, 180nM, 200nM , 250nM, 300nM, 350nM, 400nM, 450nM or 500nM.
  • the final concentration of the complementary probes of the four Aspergillus species described in step (2) is 50-500nM, for example, it can be 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 120nM, 150nM, 180nM, 200nM , 250nM, 300nM, 350nM, 400nM, 450nM or 500nM.
  • the final concentration of the specific primers for each Aspergillus in the PCR reaction system of this application is the same, according to the combination ratio of four pairs of primers 1:1:1:1, the final concentration range is the final concentration range of 50-500nM,
  • the primer probe design of this application satisfies consistent amplification efficiency.
  • the final concentration of the detection probe for each Aspergillus in the PCR reaction system of this application is the same. According to the combination ratio of the four probes at 1:1:1:1, the final concentration range is the final concentration range of 50-500nM .
  • the final concentration of the complementary probes for each Aspergillus in the PCR reaction system of this application is the same. According to the 1:1:1:1 combination ratio of the four probes, the final concentration range is the final concentration range of 50-500 nM .
  • the conditions of the real-time fluorescent PCR reaction in step (3) are:
  • step (1') also includes a preheating process: 50°C, 2 min, 1 cycle.
  • the real-time fluorescent PCR reaction in step (3) specifically includes the following steps:
  • step (3) a judgment step is further included, and the judgment standard is:
  • this application provides a primer-probe combination as described in the first aspect and/or a kit as described in the second aspect for use in Aspergillus species detection or preparation of reagents for Aspergillus species detection application.
  • the primer probe combination of the present application has high specificity and sensitivity, and can simultaneously detect Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Aspergillus terreus and distinguish specific bacterial species;
  • the detection method of the present application has high sensitivity, and can quickly detect Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Aspergillus terreus in combination with a specific primer probe combination, and can simultaneously identify and distinguish different Aspergillus species in the same fluorescence detection channel.
  • the change of the needle melting point can distinguish the specific target sequence of various Aspergillus species in the same fluorescence channel;
  • Oligonucleotide probe melting curve analysis can be carried out directly after real-time fluorescent PCR amplification reaction, without opening the tube, or it can be transferred to real-time fluorescent PCR instrument for analysis after ordinary PCR amplification;
  • Oligonucleotide probe melting curve analysis is a non-consumable test. After the analysis, the sample remains in the state after PCR, and the analysis can be repeated multiple times.
  • Figure 1 shows the melting curves of the detected products using plasmids containing DNA fragments of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Aspergillus terreus respectively as templates;
  • Example 2 is the amplification curve corresponding to the standard plasmid containing the DNA fragment of Aspergillus fumigatus at different concentrations in Example 4;
  • Example 3 is the melting curve corresponding to the standard plasmid containing the Aspergillus fumigatus DNA fragment in different concentrations in Example 4;
  • Example 4 is a graph showing the amplification curve of the Aspergillus fumigatus DNA with different concentration gradients in Example 5;
  • Fig. 5 is a graph showing the linear relationship between different concentrations of Aspergillus fumigatus DNA and Ct value in Example 5.
  • nucleotide sequences of specific primers, detection probes and complementary probes for Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Aspergillus terreus are shown in Table 1.
  • Negative control The negative control is a plasmid containing an Arabidopsis DNA fragment.
  • the nucleotide sequence of the Arabidopsis DNA fragment is shown in SEQ ID NO. 20, and the specific sequence is as follows:
  • Positive quality control products plasmids containing Aspergillus fumigatus DNA fragments, plasmids containing Aspergillus flavus DNA fragments, plasmids containing Aspergillus niger DNA fragments and plasmids containing Aspergillus terreus DNA fragments.
  • the nucleotide sequence of the Aspergillus fumigatus DNA fragment is shown in SEQ ID NO. 21, and the details are as follows:
  • the nucleotide sequence of the Aspergillus flavus DNA fragment is shown in SEQ ID NO.22, and the details are as follows:
  • the nucleotide sequence of the Aspergillus niger DNA fragment is shown in SEQ ID NO.23, and the details are as follows:
  • the nucleotide sequence of the Aspergillus terreus DNA fragment is shown in SEQ ID NO. 24, and the details are as follows:
  • Auxiliary reagents Taq enzyme, dNTP, MgCl 2 , DMSO and buffer.
  • the centrifuge tube containing the above-mentioned components and instructions are loaded into the kit.
  • Example 1 Using the kit in Example 1 to detect Aspergillus species, including the following steps:
  • step (3) Put the system of step (2) into a real-time fluorescent PCR machine to perform PCR amplification reaction.
  • the specific conditions are as follows:
  • the detection fluorescence channel of step (4") is FAM, run the PCR reaction program, and save the file;
  • the negative control group is effective: the negative control group has no typical amplification curve or no Ct value under the FAM channel, otherwise there may be reagent contamination, please remove the source of contamination and retest;
  • the positive quality control group is valid: the positive quality control group has a typical amplification curve under the FAM channel. After confirming that it is correct, adjust the Ct value of the positive quality control to 20 by adjusting the threshold, and use this as the threshold of the sample to be tested;
  • the melting temperature of the DNA sequence of different Aspergillus species is different, and the instrument is calibrated through the four plasmids of positive quality control.
  • This application uses Roche 480 for detection to obtain Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus terreus Corresponding product annealing temperature table (Table 3), it should be noted that any instrument that meets real-time fluorescent PCR is acceptable, and there is no need to make specific restrictions here.
  • the purpose of calibration is to eliminate the specific annealing temperature difference caused by different instruments.
  • Cryptococcus, Aspergillus and Candida are common clinical fungi of primer-invasive fungal diseases.
  • the above-mentioned bacteria are common species that cause bacterial infections such as respiratory tract. It can be seen from Table 4 that the detection system of the present application does not produce an amplification reaction in the presence of nucleic acid of the above-mentioned fungi or bacteria, nor can it detect a specific melting curve and Tm value, indicating that the present application has good specificity .
  • the melting curve method was used to detect the sensitivity of plasmid standards of four Aspergillus target sequences at different concentrations.
  • the synthetic target sequence plasmid standards of Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus and Aspergillus niger (Shanghai Shenggong Synthesis) were used to dilute into 10 6 copies, 10 5 copies, 10 4 copies, 10 3 copies, 10 2 copies, 10 1 copies, 10 0 copies.
  • the Roche 480II fluorescent quantitative PCR instrument was used for detection, and melting curve analysis was performed.
  • the amplification curves of different concentrations are shown in Figure 2.
  • the curves 1-8 correspond to 10 6 copies, 10 5 copies, 10 4 copies, 10 3 copies, 10 2 copies, 10 1 copies, 10 0 copies and negative control, the melting curve is shown in Figure 3.
  • the four Aspergillus species that can be detected by this kit are evenly mixed at a concentration of 1ng/ ⁇ l each, and a 10-fold serial dilution is performed to prepare a standard curve with 7 concentration points. Each concentration is performed in 3 replicate wells.
  • the gene fragment of Arabidopsis thaliana was used as a negative control, the amplification curve is shown in Figure 4, and the linear relationship between Ct value and concentration is established in Figure 5.
  • the above-mentioned standard curve, negative control, and sample DNA of unknown concentration are used for fluorescent PCR reaction.
  • Roche LightCycler 480II, ABI7500, StepOnePlus and other fluorescent quantitative PCR instruments its built-in analysis software can be based on the standard curve and the Ct of the sample. Value, calculate the nucleic acid concentration of the target gene in the sample, and perform quantitative analysis.
  • auxiliary reagents in Example 1 and the specific primers, detection probes and complementary probes of Aspergillus fumigatus in accordance with the components and dosages in the following table constitute the stability detection kit-Fumigatus; using the auxiliary in Example 1
  • the reagents and specific primers, detection probes and complementary probes of Aspergillus flavus are composed of the components and dosages in the following table.
  • Stability test kit-Aspergillus flavus using the auxiliary reagents in Example 1 and specific primers of Aspergillus terreus,
  • the detection probes and complementary probes compose the stability detection kit-Aspergillus terreus according to the components and dosages in the following table; use the auxiliary reagents in Example 1 and the specific primers, detection probes and complementary probes of Aspergillus niger according to the following
  • the components and dosages in the table constitute the stability detection kit-Aspergillus niger, and the separate detection system for each bacteria is shown in Table 5;
  • Example 2 Use the method in Example 2 to compare the Ct value of the DNA of the above-mentioned bacterial species detected by the kit in Example 1 and the Ct value of the corresponding bacterial species in the four stability test kits in 1), namely Using the experimental method in Example 2 to compare the Ct value of the four Aspergillus DNA detection in Example 1 and the Ct value of the stability test kit for the four Aspergillus DNA detection results, and analyze and compare the standards of the two groups of results Difference and CV value, the results are shown in Table 6 below;

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Abstract

La présente invention concerne une combinaison amorce-sonde pour la détection et la distinction d'espèces Aspergillus, un kit, un procédé de détection et leur utilisation. La combinaison amorce-sonde pour la détection et la distinction d'espèces Aspergillus comprend des amorces spécifiques, des sondes de détection et des sondes complémentaires pour Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger et Aspergillus terreus, respectivement. La sonde complémentaire et la sonde de détection ne sont pas complètement complémentaires et appariées. La combinaison amorce-sonde de la présente demande détermine s'il s'agit d'une infection à Aspergillus au moyen d'une détection, et peut distinguer simultanément Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger et Aspergillus terreus dans le même canal fluorescent.
PCT/CN2019/112918 2019-05-07 2019-10-24 Combinaison amorce-sonde pour la détection et la distinction d'espèces aspergillus, kit, procédé de détection et leur utilisation WO2020224195A1 (fr)

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