WO2019148590A1 - 一种检测艰难梭菌菌属基因及毒素基因的多重荧光pcr试剂盒和方法 - Google Patents

一种检测艰难梭菌菌属基因及毒素基因的多重荧光pcr试剂盒和方法 Download PDF

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WO2019148590A1
WO2019148590A1 PCT/CN2018/078623 CN2018078623W WO2019148590A1 WO 2019148590 A1 WO2019148590 A1 WO 2019148590A1 CN 2018078623 W CN2018078623 W CN 2018078623W WO 2019148590 A1 WO2019148590 A1 WO 2019148590A1
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tpi
tcdb
gene
kit
multiplex
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罗宝花
曾宏彬
陈杰
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广州赛哲生物科技股份有限公司
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Definitions

  • the present invention relates to the field of bacterial detection technology, and in particular to a multiplex fluorescent PCR kit and method for detecting a C. difficile gene and a toxin gene.
  • Clostridium difficile also known as Clostridium difficile
  • CD was first isolated from healthy neonatal feces by Hall in 1935. Its nutritional requirements are high, and conventional anaerobic culture methods are not easy to grow, and it is difficult to isolate and culture. Therefore, it is named.
  • CD is a component of the intrinsic flora in healthy human body, accounting for less than 3%. It belongs to conditional pathogens. The cause of disease depends mainly on whether it is a toxigenic strain and the amount of bacteria and the amount of toxin produced. Under normal circumstances, microorganisms in the human intestinal tract are interdependent and mutually restricted, maintaining a certain number and proportion of micro-ecological balance.
  • the normal intestinal flora has a certain ability to resist pathogens, can antagonize and limit the colonization of CD in the intestine, and degrade the toxins produced by it, so that CD does not show pathogenicity.
  • CD is resistant to antibiotics such as ampicillin, cephalosporin, lincomycin, clindamycin, erythromycin, tetracycline, etc. If long-term use of these antibiotics (especially clindamycin) or decreased immunity, Bacterial parasitic parasitism (such as gastrointestinal surgery or gastric tube insertion), repeated enema, use of anti-tumor chemicals, etc.
  • CDI Clostridium difficile infection
  • CDAD Clostridium difficile-associated diarrhea
  • CDAC Clostridium difficile-associated colitis
  • Toxins A and B secreted by C. difficile are associated with the pathogenesis of CDAD, and the two toxins are encoded by the tcdA and tcdB genes, respectively.
  • the negative regulatory gene tcdC, the forward regulatory gene tcdD, and the membranous protein gene tcdE together constitute a pathogenicity locus (PaLoc).
  • Toxin A is highly toxic to the intestine, has white blood cell (WBC) chemotaxis and certain cytotoxic effects, and increases the secretion of water and salt by mediating the cAMP system of mucosal epithelial cells, causing secretory diarrhea and even mucosal bleeding;
  • Toxin B is mainly characterized by cytotoxicity, which can stimulate the release of inflammatory factors by monocytes, causing an inflammatory reaction and causing exudative diarrhea.
  • Cases of a toxin A-negative and toxin-B positive CD strain have been clinically found to indicate that toxin B can be directly pathogenic.
  • Triose phosphate isomerase (tpi) is a metabolic enzyme secreted by C. difficile. The tpi gene is a housekeeping gene of C. difficile. It is strain-specific and can be used as a genotyping marker to replace 16S ribosomal DNA. .
  • CDI laboratory diagnosis is mainly to analyze the stool samples of patients with diarrhea, and there are many types of detection methods.
  • CD laboratory tests include cell culture toxin assay (CTA), toxin-linked immunosorbent assay (ELISA), anaerobic culture assay, lactate dehydrogenase (GDH) assay, and molecular biology assay.
  • CTA cell culture toxin assay
  • ELISA toxin-linked immunosorbent assay
  • GDH lactate dehydrogenase
  • molecular biology assay molecular biology assay.
  • the "gold standard" CTA method is difficult to operate, long in cycle, and high in cost, and has not been widely used in clinical microbiology laboratories; ELISA method has become one of the most widely used detection methods at home and abroad because of its simple and rapid operation.
  • the method has the problems of poor sensitivity and easy to miss detection; the anaerobic culture method has a long cycle and high cost, and can not directly detect the toxigenic C. difficile, so the application is less; the GDH method cannot identify whether the strain produces toxin.
  • Real-time fluorescent PCR technology has become the mainstream detection technology for C. difficile detection with its characteristics of fast, sensitive, high-throughput and automation. Compared with other technical methods, it has the advantages of wide linear range and low risk of closed-tube pollution.
  • existing fluorescent PCR techniques still have false negatives or false positives, and the lack of accuracy, stability and sensitivity limit their clinical application.
  • the sample to be tested contains complex components (such as whole blood, easy to stop, diarrhea, metronidazole or vancomycin, etc.), the accuracy and sensitivity of the existing kits for CD detection are often not ideal. .
  • the object of the present invention is to overcome the above-mentioned deficiencies of the prior art and to provide a set of multiplex PCR primer sets and probes for detecting C. difficile gene and toxin gene, which can be designed by designing specific primer sets and probes.
  • the C. difficile gene and toxin gene were detected in the complex samples, which were simple in operation, short in reporting time, strong in specificity, high in sensitivity and good in accuracy.
  • the internal standard was introduced to avoid PCR false negatives, which reduced the amplification products. False positive caused by pollution.
  • Another object of the present invention is to provide the use of the above multiplex fluorescent PCR primer set and probe for the preparation of multiplex fluorescent PCR reagents or kits for detecting C. difficile genes and toxin genes.
  • Another object of the present invention is to provide a multiplex fluorescent PCR kit for detecting a C. difficile gene and a toxin gene, and the fecal specimen detected by the kit can simultaneously complete C. difficile in a short time without pure culture. Identification of the genus and detection of the toxin carrying of the strain.
  • Another object of the present invention is to provide a multiplex fluorescent PCR method for detecting a Clostridium gene and a toxin gene.
  • a set of multiplex PCR primer sets and probes for detection of C. difficile genes and toxin genes including tpi amplification primer pairs tpi-F and tpi-R, tpi fluorescent probe tpi-P, tcdB amplification primer pair tcdB -F and tcdB-R, tcdB fluorescent probe tcdB-P, internal standard primer pair HP-F and HP-R, internal standard fluorescent probe HP-P; nucleotide sequence is SEQ ID NO: 1 - SEQ ID NO:9.
  • the present invention adopts multiplex fluorescent PCR combined with specific primers and Taqman probe technology to design both tpi and tcdB specific primers and probes to identify both C. difficile and toxin B genes in a PCR reaction. the goal of.
  • internal standards abbreviated as internal standards
  • the specific method is as follows: Firstly, the target fragments derived from other species of the C. difficile genome are selected as alternative internal standards by sequence alignment in the Genebank database.
  • the candidate internal standard and the kit tpi, tcdB targets are subjected to PCR detection one by one, and finally HP is preferably used as an internal standard.
  • HP is preferably used as an internal standard.
  • the probe has a fluorescent substance bound to the 5' end and a quenching substance bound to the 3' end; the fluorescent substance is at least one of FAM, HEX, TET, JOE, VIC, Cy5; The extinction substance is at least one of TAMRA, BHQ1, BHQ2, and BHQ3.
  • the fluorescent substance bound to the 5' end of the probe is at least one of FAM, HEX, and Cy5, and the fluorescent substance bound at the 3' end is BHQ1.
  • the fluorescent substances bound to the 5' end of the probes tpi-P, tcdB-P, and HP-P are FAM, HEX, and Cy5, respectively.
  • the present invention also claims the use of any of the multiplex fluorescent PCR primer sets and probes described above for the preparation of multiplex fluorescent PCR reagents or kits for detecting C. difficile genes and toxin genes.
  • the invention also claims the use of any of the multiplex fluorescent PCR primer sets and probes described above for the preparation of a reagent or kit for detecting a C. difficile gene and a toxin gene in a complex sample.
  • the complex sample refers to at least one impurity containing whole blood, easy to stop, diarrhea, metronidazole or vancomycin in the sample to be tested.
  • the present invention also claims a multiplex fluorescent PCR kit for detecting a C. difficile gene and a toxin gene, comprising any of the multiplex fluorescent PCR primer sets and probes described above.
  • the kit can be used to detect C. difficile genes and toxin genes in the above complex samples, and therefore, the kit is a kit for detecting C. difficile genes and toxin genes in complex samples.
  • tpi-F, tpi-R, tpi-P, tcdB-F, tcdB-R, tcdB-P, HP-F, HP-R, HP-P The final concentration ratio is 3:3:2:4:4:2.5:2:2:1.5.
  • the multiplex PCR reaction conditions of the kit are: 37 ° C for 5 min; 95 ° C for 5 min; 95 ° C for 15 s, 60 ° C for 38 s, 45 cycles.
  • the kit further comprises a nucleic acid extraction reagent, a reagent for multiplex PCR reaction, an internal standard working solution, a positive control solution and a negative control solution;
  • the nucleic acid extraction reagent comprises a lysate, a washing buffer I, a washing buffer Liquid II and DNA eluate;
  • reagents required for the multiplex fluorescent PCR reaction include MgCl 2 , 10 ⁇ PCR buffer, dNTP/dUTP mixed solution, UDG enzyme and Taq DNA polymerase;
  • the internal standard working solution is tpi-free And the HP plasmid of the amplified sequence of the tcdB gene;
  • the positive control solution is a cloning plasmid containing the amplified sequence of the tpi and tcdB genes; and the negative control solution is sterilized ultrapure water.
  • the kit of the invention increases the internal standard quality control system, monitors whether the sample to be tested contains PCR inhibitors and evaluates the quality of nucleic acid extraction, avoids PCR false negative; and uses a cloned plasmid containing the amplified sequences of tpi and tcdB genes as a positive control
  • the solution by adding UDG enzyme + dUTP anti-pollution system, fully degrades the possible amplification product contamination, avoiding false positive results.
  • the lysate comprises SDS, trisodium citrate dihydrate, DTT and glycogen.
  • the invention also claims a multiplex PCR method for detecting a C. difficile gene and a toxin gene, comprising the steps of:
  • the fluorescent signal is collected, and the result is analyzed by measuring the Ct value of the DNA.
  • the present invention has the following beneficial effects:
  • the kit of the invention has an internal standard quality control system, and three targets of tpi gene, tcdB gene and internal standard can be detected at one time in the same reaction tube.
  • tpi gene the identification of the genus Clostridium can be carried out;
  • tcdB gene it can be simultaneously judged whether the strain to be tested carries the gene of toxin B, and is effectively used for judging the secretion of the clinical strain and its toxin.
  • the internal standard system can monitor whether the sample to be tested contains PCR inhibitors and evaluate the quality of nucleic acid extraction, and avoid PCR false negatives.
  • the kit of the invention also adopts a cloning plasmid containing the amplified sequences of tpi and tcdB genes as a positive control solution, and fully degrades the possible amplification product contamination by adding a UDG enzyme + dUTP anti-pollution system to avoid false positive results.
  • the kit of the invention enables the fecal specimen to be extracted without pure culture, and has the advantages of simple operation, short reporting time, strong specificity, high sensitivity and good accuracy, and strong anti-interference ability.
  • the C. difficile gene and toxin gene were detected from complex samples, which were suitable for the screening of etiology of patients with unexplained diarrhea.
  • Figure 1 is the genomic DNA related data (concentration and OD value) of the stool sample in Example 2.
  • Example 2 is a positive control diagram of the quality control C. difficile toxigenic strain in Example 2.
  • Figure 3 is a graph showing the positive control of the quality control C. difficile non-toxigenic strain in Example 2.
  • Figure 4 is a negative control diagram of the quality control C. difficile in Example 2.
  • Figure 5 is a cross-reactivity analysis diagram of specificity analysis in Example 3.
  • Figure 6 is a graph showing the interference analysis of endogenous substances for specific analysis in Example 3.
  • Figure 7 is a graph showing the interference analysis of exogenous substances specifically analyzed in Example 3.
  • Fig. 8 is a graph showing a triple fluorescent PCR amplification curve of Clostridium difficile at a concentration of 3.0 ⁇ 10 2 bacteria/mL in Example 3.
  • Figure 9 is a double fluorescence amplification curve of the control kit A for detecting C. difficile.
  • Figure 10 is a double fluorescence amplification curve of a control kit B for detecting C. difficile.
  • Figure 11 is a double fluorescence amplification curve of a control kit C for detecting C. difficile.
  • Figure 12 is a double fluorescence amplification curve of the kit of Example 1 for detecting C. difficile.
  • Figure 13 is a triple fluorescence amplification curve of a control kit C for detecting C. difficile.
  • Figure 14 is a triple fluorescence amplification curve of the kit of Example 1 for detecting C. difficile.
  • test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used are, if not specified, commercially available reagents and materials.
  • a multiplex PCR kit for detecting C. difficile gene and toxin gene comprising a set of multiplex PCR primer sets and probes for detecting C. difficile gene and toxin gene, nucleic acid extraction reagent, multiplex PCR reaction Required reagent, internal standard working solution, positive control solution and negative control solution.
  • the multiplex PCR primer set and probe for detecting a Clostridium gene and a toxin gene including tpi amplification primer pair tpi-F and tpi-R, tpi fluorescent probe tpi-P, tcdB amplification primer
  • tcdB-F and tcdB-R tcdB fluorescent probe tcdB-P
  • HP-F and HP-R internal standard fluorescent probe HP-P
  • nucleotide sequence is SEQ ID NO: 1 to SEQ ID NO: 9.
  • the fluorescent substances bound to the 5' end of the probes tpi-P, tcdB-P, and HP-P were FAM, HEX, and Cy5, respectively, and the fluorescent substance bound at the 3' end was BHQ1.
  • the final concentration of tpi-F, tpi-R, tpi-P, tcdB-F, tcdB-R, tcdB-P, HP-F, HP-R, HP-P in the multiplex PCR reaction system of the kit The ratio is 3:3:2:4:4:2.5:2:2:1.5.
  • the multiplex PCR reaction conditions of the kit were: 37 ° C for 5 min; 95 ° C for 5 min; 95 ° C for 15 s, 60 ° C for 38 s, 45 cycles.
  • the nucleic acid extraction reagent comprises a lysate, a washing buffer I, a washing buffer II and a DNA eluent;
  • the reagents required for the multiplex fluorescent PCR reaction include MgCl 2 , 10 ⁇ PCR buffer, dNTP/dUTP mixed solution, UDG enzyme And the Taq DNA polymerase;
  • the internal standard working solution is an HP plasmid containing no amplification sequence of the tpi and tcdB genes;
  • the positive control solution is a cloning plasmid containing the amplified sequences of the tpi and tcdB genes;
  • the negative control solution is Sterilize ultrapure water.
  • the lysate includes SDS, trisodium citrate dihydrate, DTT, and glycogen.
  • the method for using the kit comprises the steps of: extracting clinical sample fecal genomic DNA and negative control DNA, and adding an internal standard working solution to the lysate; adding a PCR reaction to the sample to be tested, the negative control group and the positive control group
  • the system uses the kit for multiplex PCR reaction; collects the fluorescent signal, and analyzes the Ct value of the DNA to analyze the result.
  • the preparation process of the kit is as follows:
  • sequences of other source species that are not homologous to the C. difficile genomic sequence were selected as internal standards.
  • the internal standard can be used to control the quality of the false negative results caused by inhibitors, instruments, reagents and operations that may exist during the experiment.
  • the internal standard should be designed to ensure that the additional internal standard fluorescent channel is clearly positive when it is detected, and that the inhibition of the target nucleic acid sequence detection is minimized to cause false negatives.
  • HP is finally recommended as an internal standard (commissioned by Shanghai Jierui Bioengineering Co., Ltd. to synthesize HP amplified region fragments and inserted into pGHn plasmid vector).
  • the Genebank serial number of the tpi gene, tcdB gene and internal standard sequence is shown in Table 1:
  • the full sequence of the primer design template CP011968.1 was aligned in NCBI blast, and the sequence with higher specificity was determined. Using this part of the sequence as a primer design template, three pairs of primer sets were designed. Table 2 shows. The GC content, Tm value, hairpin structure and primer mismatch frequency of the primer probe were analyzed to screen out the best tpi primers and probes.
  • primers and probes for tcdB and HP were designed according to the tpi primer and probe design methods.
  • Preferred primers and probe information for tpi, tcdB, and HP are shown in Table 3.
  • the nucleic acid extraction reagent is used to extract fecal sample genomic DNA, consisting of lysate, washing buffer I, washing buffer II and DNA eluate.
  • the lysate is prepared by SDS, trisodium citrate dihydrate, glycogen, DTT, etc.; washing buffer I includes Tris-HCl buffer, EDTA, sodium chloride, and washing buffer II includes Tris-HCl buffer. , sodium chloride, DNA eluate including Tris-HCl buffer, EDTA.
  • the multiplex fluorescent PCR reaction system includes the primer set and probe in Table 3, and 50 mM MgCl 2 , 10 ⁇ PCR buffer, dNTP/dUTP mixed solution, UDG enzyme, and Taq DNA polymerase, and pathogen detection was performed by PCR in vitro amplification.
  • the final concentration ratio of primers and probes tpi-F, tpi-R, tpi-P, tcdB-F, tcdB-R, tcdB-P, HP-F, HP-R, HP-P was 3:3 in order. :2:4:4:2.5:2:2:1.5.
  • the positive control solution was prepared from a plasmid containing the tpi gene and the tcdB gene, and its concentration was measured using an ultraviolet spectrophotometer. After dilution, it was used as a positive control product of the kit; the negative control solution was sterilized ultrapure water.
  • the internal standard working solution is a synthetic plasmid, which does not contain the amplified sequences of tpi and tcdB genes. It was commissioned by Shanghai Jierui Bioengineering Co., Ltd. to synthesize the internal standard amplified region fragment (HP) and inserted into the pGHn plasmid vector according to the UV spectrophotometer. The OD value was measured to calculate the copy number, and after the gradient dilution, it was used as the internal standard working solution of the kit.
  • the C. difficile in the clinical sample was detected using the kit of Example 1, and the specific steps are as follows:
  • Collect human stool samples which should not be mixed with urine, toilet water, disinfectant, should not touch the toilet bowl, toilet paper, diaper, etc., in order to prevent the sample from being contaminated or destroyed.
  • Set 10 C. difficile positive clinical samples P1-P10) and 10 C. difficile negative clinical samples (N1-N10), including positive C. difficile-producing strains (P1-P5) and non-toxigenic strains (P1-P5) P6-P10). All clinical stool samples were verified by isolation culture, mass spectrometry identification and toxin Sanger sequencing of the General Hospital of Guangzhou Military Region.
  • the DNA was eluted by centrifugation at 12,000 rpm for 1 min.
  • the extracted nucleic acid should be used immediately for subsequent detection. If it cannot be detected in time, it should be stored at -20 °C.
  • Genomic DNA is extracted from the sample, and an internal standard is added to the clinical sample to participate in and monitor the nucleic acid extraction process.
  • the relevant data (concentration and OD value) of genomic DNA in the extracted stool samples are shown in Fig. 1. From the results, the purity of the extracted genomic DNA was within the ideal range (OD 260/280 : 1.8 to 2.0); the DNA concentration ranged from 10 to 350 ng/ ⁇ L due to clinical sample variability.
  • the PCR reaction system was added to the sample to be tested, the negative control group and the positive control group, and mixed, and after transient centrifugation, the sample was dispensed into the PCR reaction tube at 17 ⁇ L/tube; the respective reaction tubes were separately added and processed. 3 ⁇ L of DNA sample, 3 ⁇ L of negative control solution and 3 ⁇ L of positive control solution. Centrifuge briefly to concentrate all reagents on the bottom of the reaction tube (no air bubbles), and immediately after the cap is closed, perform a PCR amplification reaction.
  • Fluorescence detection channel selection select FAM channel to detect tpi; select HEX/VIC channel to detect tcdB; select Cy5 channel to detect internal standard.
  • the quenching groups are selected as None.
  • Negative control The amplification curve of FAM detection channel and HEX detection channel has no logarithmic growth period, and the amplification curve of Cy5 detection channel is logarithmic growth period, and Ct value is ⁇ 36;
  • the amplification curve of FAM detection channel and HEX detection channel has a significant logarithmic growth period, and Ct ⁇ 24.
  • the detection results of the clinical sample P1-P10 of the kit of Example 1 are the same as those of the anaerobic culture mass spectrometry and the toxin sanger sequencing; and the anaerobic culture method is identified as the C. difficile negative 10
  • the kit of Example 1 detected that N1-N10 was negative for C. difficile tpi and tcdB. Therefore, the result of detecting the C. difficile in the kit of Example 1 is consistent with the expected result, and the operation is convenient and quick, and is not easily affected by the environment.
  • Example 2 Using the kit described in Example 1 and the detection method described in Example 2, selecting microorganisms similar to or causing similar symptoms to C. difficile species, such as Yersinia enterocolitica, enterotoxic Escherichia coli and waxy 21 species such as Bacillus were used as samples to be tested, and the conformity of the kit of Example 1 to other pathogens causing similar symptoms of Clostridium difficile was evaluated to analyze the specificity of the kit.
  • the OD value of the sample to be tested is estimated to be higher than 1 ⁇ 10 6 bacteria/mL, and the results are shown in Table 7 and Figure 5.
  • Example 2 Using the kit described in Example 1 and the detection method described in Example 2, 200 mg of feces was added to 200 ⁇ L of C. difficile inactivated culture (7.88 ⁇ 10 4 bacteria/mL) for detection, and the same amount of difficulty was taken.
  • the Clostridium inactivating culture was a positive control, and it was evaluated whether the sample containing the endogenous substance had an influence on the sensitivity of the kit to analyze the interference of these endogenous substances, and the results are shown in Table 8 and Figure 6.
  • the experimental results show that under the experimental conditions of this kit, the Ct value variation coefficient is less than 5% after the C. difficile inactivated culture is added to the feces, and the endogenous substances that may exist in the sample do not interfere with the detection results of the kit. .
  • Example 2 Using the kit described in Example 1 and the detection method described in Example 2, the following interfering substances were added to the C. difficile inactivated culture sample at a concentration of 7.88 ⁇ 10 5 bacteria/mL (P1), and An equal amount of C. difficile inactivated culture was used as a positive control. It was evaluated whether the sample containing exogenous substances had an influence on the sensitivity of the kit to investigate the interference of these exogenous substances. The results are shown in Table 9 and Figure 7.
  • the experimental results show that the Ct value variation coefficient of the kit is less than 5% after the addition of other therapeutic drugs to the C. difficile culture, indicating that the exogenous interfering substance pair reagent may exist in the sample under the experimental conditions of the kit.
  • the test results of the box are free from interference.
  • the minimum concentration of bacteria detected at 100% is between 2.62 ⁇ 10 2 bacteria / mL and 7.88 ⁇ 10 2 bacteria / mL.
  • the bacterial sample having the above-mentioned estimated minimum detection limit concentration was used as an initial sample, and after three-fold dilution, the samples were examined using the kit described in Example 1 and the kit detection method described in Example 2. For each concentration, 20 replicates were made for each batch of kits to analyze the detection rate of each concentration sample. The lowest concentration detected above 95% is the detection limit.
  • the experimental results showed that the positive detection rate of C. difficile at a concentration of 3.0 ⁇ 10 2 bacteria/mL was 100%, as shown in FIG. 8 . This indicates that the minimum detection limit of the kit is 3.0 ⁇ 10 2 bacteria / mL.
  • Set control kits A, B and C select animal virus sequences as internal standards, respectively HCS, HJ and HSa, add synthetic plasmids to the sample to participate in the nucleic acid extraction process, respectively design primers, probes, in addition to internal standard
  • the pair of particles and the internal standard fluorescent probe were different, and the remaining components were identical to the kit of Example 1. Since the amplification efficiency of tpi is much higher than that of tcdB, only tcdB and internal standard were selected for double fluorescent PCR detection, and the rest was consistent with the detection method of Example 2. Clinical samples were separately tested using the kit of Example 1 and the control kits A, B, and C, with four template concentration gradients set for each kit.
  • control kit C and the kit of Example 1 with better amplification curves in the dual fluorescence, the clinical samples were detected by the triple fluorescent PCR using the detection method of Example 2, and four template concentration gradients were set.
  • the control kit D was set up except that there was no internal standard primer pair HP-F and HP-R, the internal standard fluorescent probe HP-P, and the remaining components were identical to the kit of Example 1.
  • the standard strain ATCC9689 of C. difficile (the strain was diluted 10 times by 10 times) and the specific strains N1 to N10 were respectively determined, and the standard strain ATCC9689 was judged. Whether the Ct values of the tpi and tcdB targets of the specific strains were changed, and the results are shown in Table 11.
  • the Ct value of the standard strain ATCC9689 is substantially the same, almost unchanged, thereby indicating that the internal standard does not inhibit the amplification of the tpi and tcdB targets.
  • the tpi and tcdB targets of the specific strain failed to expand, indicating that the addition of the internal standard of the kit of Example 1 did not result in a false positive.

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Abstract

本发明公开了一种检测艰难梭菌菌属基因及毒素基因的多重荧光PCR试剂盒和方法，主要包括特异性引物组和探针，所述引物组和探针由检测艰难梭菌的引物对和探针、检测毒素B的引物组和探针、内标引物对和探针组成。

Description

一种检测艰难梭菌菌属基因及毒素基因的多重荧光PCR试剂盒和方法 技术领域
本发明涉及细菌检测技术领域，具体地，涉及一种检测艰难梭菌菌属基因及毒素基因的多重荧光PCR试剂盒和方法。
背景技术
艰难梭菌(Clostridium difficile,CD)又称难辨梭状芽孢杆菌，由Hall等于1935年首次从健康新生儿粪便中分离，其营养要求较高，常规厌氧培养法不易生长，分离培养较为困难，故因此得名。CD为健康人体中固有菌群的组成部分，约占3％以下，属于条件致病菌，致病与否主要取决于是否为产毒菌株以及细菌数量和产毒量。正常情况下，人体肠道中微生物之间相互依赖、又彼此制约，维持着一定数量和比例的微生态平衡。肠道正常菌群具有一定的抵御病原体的能力，可拮抗和限制CD在肠道内的定殖，并降解其产生的毒素，使得CD不表现致病性。CD对氨节西林、头孢菌素、林可霉素、克林霉素、红霉素、四环素等抗生素耐药，若长期使用这些抗生素(尤其是克林霉素)或出现机体免疫力降低、菌体移位寄生(如胃肠道外科手术或胃管插入)、反复灌肠、使用抗肿瘤化学制剂等情况时均可影响肠道内微生态平衡，引起菌群失调，肠道内的双歧杆菌、乳杆菌等优势菌群对CD的拮抗作用受到限制，耐药的CD大量繁殖而致病，易引起艰难梭菌感染(Clostridium difficile infection,CDI)，进而引起艰难梭菌相关性腹泻(Clostridium difficile-associated diarrhea,CDAD)和艰难梭菌相关性肠炎(Clostridium difficile-associated colitis,CDAC)，甚至严重者有致死性假膜性肠炎和中毒性巨结肠，临床多以肠道炎性病变和形成伪膜为主要特点。
艰难梭菌分泌的毒素A和B与CDAD发病有关，2个毒素分别由tcdA和tcdB基因编码。此外，负向调节基因tcdC、正向调节基因tcdD以及膜孔蛋白基因tcdE共同构成致病性决定区(Pathogenicity Locus,PaLoc)。毒素A对肠道有较强的毒性，有白细胞(WBC)趋化和一定的细胞毒性作用，通过介导黏膜上皮细胞的cAMP系统使水和盐分泌增加，引发分泌性腹泻，甚至黏膜出血；毒素B主要表现为细胞毒性，可刺激单核细胞释放炎性因子，引起炎性反应而引发渗出性腹泻。临床上已发现毒素A阴性而毒素B阳性的CD菌株感染的病例，表明毒素B可单独直接致病。磷酸丙糖异构酶(tpi)是艰难梭菌分泌的一种代谢酶，tpi基因是艰难梭菌的管家基因，具有菌种特异性，可作为一种替代16S核糖 体DNA的基因分型标志。
CDI实验室诊断主要是分析腹泻患者的粪便标本，检测方法种类多。目前CD实验室检查有细胞培养毒素检测法(CTA)、毒素酶联免疫吸附试验(ELISA)、厌氧培养法、乳酸脱氢酶(GDH)检测、分子生物学检测等。但是，“金标准”CTA法操作难、周期长、成本高，尚未广泛应用于临床微生物实验室；ELISA法因操作简便、快速，而成为目前国内外广泛最应用的检测方法之一，但该方法存在敏感性差、容易漏检的问题；厌氧培养法周期长、成本高，且无法直接检测产毒性艰难梭菌，因此应用较少；GDH法则不能鉴别菌株是否产毒。实时荧光PCR技术以其快速、灵敏、高通量和自动化的特点，逐渐成为艰难梭菌检测的主流检测技术，与其他技术方法相比，其具有线性范围广、闭管污染风险小等优点。但是，现有荧光PCR技术仍然存在假阴性或假阳性，准确性、稳定性和灵敏度低等不足，限制了其在临床上的应用。此外，当待检测样本中含有复杂成分(如全血、易蒙停、泻立停、甲硝唑或万古霉素等)时，现有试剂盒对CD检测的准确性和灵敏度往往不甚理想。
发明内容
本发明的目的是为了克服现有技术的上述不足，提供了一组检测艰难梭菌菌属基因及毒素基因的多重荧光PCR引物组和探针，通过设计特异性引物组和探针，能从成分复杂的样本中检测出艰难梭菌菌属基因及毒素基因，操作简单、报告时间短，特异性强、灵敏度高、准确性好，同时引入内标避免PCR假阴性，降低了由于扩增产物污染而导致的假阳性。
本发明的另一个目的是提供上述多重荧光PCR引物组和探针在制备检测艰难梭菌菌属基因及毒素基因的多重荧光PCR试剂或试剂盒方面的应用。
本发明的另一个目的是提供一种检测艰难梭菌菌属基因及毒素基因的多重荧光PCR试剂盒，采用该试剂盒检测的粪便标本无需纯培养，即可在短时间内同时完成艰难梭菌菌属鉴定及菌株毒素携带情况的检测。
本发明的另一个目的是提供一种检测艰难梭菌菌属基因及毒素基因的多重荧光PCR方法。
为了实现上述目的，本发明是通过以下方案予以实现的：
一组检测艰难梭菌菌属基因及毒素基因的多重荧光PCR引物组和探针，包括tpi扩增引物对tpi-F和tpi-R，tpi荧光探针tpi-P，tcdB扩增引物对tcdB-F和tcdB-R，tcdB荧光探针tcdB-P，内标引物对HP-F和HP-R，内标荧光探针HP-P；核苷酸序列分别依次如SEQ  ID NO:1～SEQ ID NO:9所示。
本发明采用多重荧光PCR并结合特异性引物和Taqman探针技术，通过设计tpi和tcdB的特异性引物和探针，实现在一个PCR反应中既能鉴定艰难梭菌菌属又能检测毒素B基因的目的。另外，设置了内标准品(简称内标)，内标可对实验过程中可能存在的扩增反应抑制物、仪器、试剂和操作等所导致的假阴性结果进行质量控制。具体方法是：首先在Genebank数据库中通过序列比对，筛选出与艰难梭菌基因组较特异的其他物种来源的靶片段作为备选内标。接着，逐个将备选内标和试剂盒tpi、tcdB靶标进行PCR检测，最后优选HP作为内标。通过对优选出的内标进行引物、探针和模板浓度优化，做到了既保证内标荧光通道呈明显的阳性曲线，又尽量降低对靶核酸序列检测造成的抑制而导致假阴性。
优选地，所述探针的5’末端结合有荧光物质，3’末端结合有淬灭物质；所述荧光物质为FAM、HEX、TET、JOE、VIC、Cy5中的至少一种；所述淬灭物质为TAMRA、BHQ1、BHQ2、BHQ3中的至少一种。
由于FAM、HEX和Cy5这3种荧光物质的波长距离相对比较大，使它们之间的相互干扰较小，提高了试剂盒的灵敏度。优选地，所述探针的5’末端结合的荧光物质为FAM、HEX、Cy5中的至少一种，3’末端结合的荧光物质为BHQ1。
更优选地，所述探针tpi-P、tcdB-P、HP-P的5’末端结合的荧光物质分别为FAM、HEX和Cy5。
本发明还请求保护上述任一所述多重荧光PCR引物组和探针在制备检测艰难梭菌菌属基因及毒素基因的多重荧光PCR试剂或试剂盒方面的应用。
本发明还请求保护上述任一所述多重荧光PCR引物组和探针在制备用于检测复杂样本中艰难梭菌菌属基因及毒素基因的试剂或试剂盒方面的应用。所述复杂样本是指待测样品中含有全血、易蒙停、泻立停、甲硝唑或万古霉素中的至少一种杂质。
本发明还请求保护一种检测艰难梭菌菌属基因及毒素基因的多重荧光PCR试剂盒，包含上述任一所述多重荧光PCR引物组和探针。
同样地，该试剂盒可用于检测上述复杂样本中的艰难梭菌菌属基因及毒素基因，因此，所述试剂盒为一种检测复杂样本中艰难梭菌菌属基因及毒素基因的试剂盒。
优选地，所述试剂盒的多重荧光PCR反应体系中，tpi-F、tpi-R、tpi-P、tcdB-F、tcdB-R、tcdB-P、HP-F、HP-R、HP-P的终浓度比依次为3：3：2：4：4：2.5：2：2：1.5。
优选地，所述试剂盒的多重荧光PCR反应条件为：37℃5min；95℃5min；95℃15s， 60℃38s，45个循环。
优选地，所述试剂盒还包括核酸提取试剂、多重荧光PCR反应所需试剂、内标工作液、阳性对照液和阴性对照液；所述核酸提取试剂包括裂解液、洗涤缓冲液I、洗涤缓冲液II和DNA洗脱液；所述多重荧光PCR反应所需试剂包括MgCl 2、10×PCR buffer、dNTP/dUTP混合溶液、UDG酶和Taq DNA聚合酶；所述内标工作液为不含tpi及tcdB基因扩增序列的HP质粒；所述阳性对照液为含有tpi及tcdB基因扩增序列的克隆质粒；所述阴性对照液为灭菌超纯水。
本发明试剂盒增加了内标质控系统，监测待测样本中是否含有PCR抑制物及评价核酸提取的质量，避免PCR假阴性；并采用含有tpi及tcdB基因扩增序列的克隆质粒作为阳性对照液，通过加入UDG酶+dUTP防污染系统将可能的扩增产物污染充分降解，避免假阳性结果。
优选地，所述裂解液包括SDS、二水柠檬酸三钠、DTT和糖原。
本发明还请求保护一种检测艰难梭菌菌属基因及毒素基因的多重荧光PCR方法，包括以下步骤：
S1.提取临床样本粪便基因组DNA和阴性对照组DNA，并向裂解液中加入内标工作液；
S2.向待测样本、阴性对照组和阳性对照组中加入PCR反应体系；
S3.采用上述任一所述试剂盒进行多重荧光PCR反应；
S4.收集荧光信号，通过测定DNA的Ct值，进行结果分析。
与现有技术相比，本发明具有以下有益效果：
(1)本发明试剂盒增加了内标质控系统，在同一反应管里可一次性检测tpi基因、tcdB基因和内标三个靶标。通过对tpi基因的检测，可以进行艰难梭菌菌属的鉴定；通过对tcdB基因的检测，可同时判断待检菌株是否携带毒素B的基因，进而有效地应用于判断临床菌株及其毒素分泌情况，提高准确性；同时内标系统可监测待测样本中是否含有PCR抑制物及评价核酸提取的质量，避免PCR假阴性。本发明试剂盒还采用含有tpi及tcdB基因扩增序列的克隆质粒作为阳性对照液，通过加入UDG酶+dUTP防污染系统将可能的扩增产物污染充分降解，避免假阳性结果。
(2)本发明试剂盒使粪便标本无需纯培养即可提取DNA进行检测，具有操作简单、报告时间短，特异性强、灵敏度高、准确性好的优点，有较强的抗干扰能力，能从成分复杂的样本中检测出艰难梭菌菌属基因及毒素基因，适用于临床不明原因腹泻病人的病因筛 查。
附图说明
图1为实施例2中粪便样品的基因组DNA相关数据(浓度及OD值)。
图2为实施例2中质量控制艰难梭菌产毒株阳性对照图。
图3为实施例2中质量控制艰难梭菌非产毒株阳性对照图。
图4为实施例2中质量控制艰难梭菌阴性对照图。
图5为实施例3中特异性分析的交叉反应分析图。
图6为实施例3中特异性分析的内源物质干扰性分析图。
图7为实施例3中特异性分析的外源物质干扰性分析图。
图8为实施例3中艰难梭菌浓度为3.0×10 2个菌/mL的三重荧光PCR扩增曲线图。
图9为对照组试剂盒A检测艰难梭菌的双重荧光扩增曲线。
图10为对照组试剂盒B检测艰难梭菌的双重荧光扩增曲线。
图11为对照组试剂盒C检测艰难梭菌的双重荧光扩增曲线。
图12为实施例1试剂盒检测艰难梭菌的双重荧光扩增曲线。
图13为对照组试剂盒C检测艰难梭菌的三重荧光扩增曲线。
图14为实施例1试剂盒检测艰难梭菌的三重荧光扩增曲线。
具体实施方式
下面结合说明书附图及具体实施例对本发明作出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例1
一种检测艰难梭菌菌属基因及毒素基因的多重荧光PCR试剂盒，包括一组检测艰难梭菌菌属基因及毒素基因的多重荧光PCR引物组和探针，核酸提取试剂，多重荧光PCR反应所需试剂，内标工作液，阳性对照液和阴性对照液。
其中，所述检测艰难梭菌菌属基因及毒素基因的多重荧光PCR引物组和探针，包括tpi扩增引物对tpi-F和tpi-R，tpi荧光探针tpi-P，tcdB扩增引物对tcdB-F和tcdB-R，tcdB荧光探针tcdB-P，内标引物对HP-F和HP-R，内标荧光探针HP-P；核苷酸序列分别依次如SEQ ID NO:1～SEQ ID NO:9所示。
所述探针tpi-P、tcdB-P、HP-P的5’末端结合的荧光物质分别为FAM、HEX、Cy5，3’ 末端结合的荧光物质均为BHQ1。
所述试剂盒的多重荧光PCR反应体系中，tpi-F、tpi-R、tpi-P、tcdB-F、tcdB-R、tcdB-P、HP-F、HP-R、HP-P的终浓度比依次为3：3：2：4：4：2.5：2：2：1.5。
所述试剂盒的多重荧光PCR反应条件为：37℃5min；95℃5min；95℃15s，60℃38s，45个循环。
所述核酸提取试剂包括裂解液、洗涤缓冲液I、洗涤缓冲液II和DNA洗脱液；所述多重荧光PCR反应所需试剂包括MgCl 2、10×PCR buffer、dNTP/dUTP混合溶液、UDG酶和Taq DNA聚合酶；所述内标工作液为不含tpi及tcdB基因扩增序列的HP质粒；所述阳性对照液为含有tpi及tcdB基因扩增序列的克隆质粒；所述阴性对照液为灭菌超纯水。所述裂解液包括SDS、二水柠檬酸三钠、DTT和糖原。
所述试剂盒的使用方法包括以下步骤：提取临床样本粪便基因组DNA和阴性对照组DNA，并向裂解液中加入内标工作液；向待测样本、阴性对照组和阳性对照组中加入PCR反应体系；采用该试剂盒进行多重荧光PCR反应；收集荧光信号，通过测定DNA的Ct值，进行结果分析。
所述试剂盒的制备过程如下：
1、引物和探针的设计
通过NCBI数据库和http://blast.ncbi.nlm.nih.gov/Blast.cgi在线比对，找出tpi基因和tcdB基因在不同艰难梭菌菌株间的保守序列。然后，通过oligo7软件，对各个基因序列进行引物探针的设计，并分析引物探针的GC含量、Tm值、发夹结构、引物错配频率等因素，研究不同引物和探针对样品的检测情况，优选出最佳的引物与探针组合。
另外，在Genebank数据库中，选择与艰难梭菌基因组序列没有同源的其它来源物种序列，作为内标。内标可对实验过程中可能存在的扩增反应抑制物、仪器、试剂和操作等所导致的假阴性结果进行质量控制。内标的设计须保证在检测时额外添加的内标荧光通道呈明显的阳性曲线，又要尽量降低其对靶核酸序列检测造成的抑制而导致假阴性。通过对内标序列的筛选及其引物、探针用量的优化，最后优选HP为内标(委托上海捷瑞生物工程有限公司合成HP扩增区域片段，并插入到pGHn质粒载体上)。tpi基因、tcdB基因和内标序列的Genebank序列号如表1所示：
表1 tpi基因、tcdB基因和内标序列的Genebank序列号
名称 登录号 位置
tpi CP011968.1 3540163-3540906
tcdB KC292135.1 3614-3833
HP KT022072.1 2833-3032
以tpi引物和探针设计为例：将引物设计模板CP011968.1全序列在NCBI blast比对，确定了特异性较高的序列，以这部分序列为引物设计模板，设计3对引物组，如表2所示。分析引物探针的GC含量、Tm值、发夹结构、引物错配频率等，筛选出检测效果最佳的tpi引物和探针。
表2 tpi引物和探针的筛选序列信息
Figure PCTCN2018078623-appb-000001
同理，按照tpi引物和探针设计方法，设计tcdB及HP的引物与探针。优选出的tpi、tcdB、HP的引物和探针信息如表3所示。
表3 试剂盒引物和探针序列表
Figure PCTCN2018078623-appb-000002
2、核酸提取试剂配制
核酸提取试剂用于提取粪便样本基因组DNA，由裂解液、洗涤缓冲液I、洗涤缓冲液II和DNA洗脱液组成。其中，裂解液由SDS、二水柠檬酸三钠、糖原、DTT等配制而成；洗涤缓冲液I包括Tris-HCl缓冲液、EDTA、氯化钠，洗涤缓冲液II包括Tris-HCl缓冲液、氯化钠，DNA洗脱液包括Tris-HCl缓冲液、EDTA。
3、多重荧光PCR反应体系配置
多重荧光PCR反应体系包括表3中的引物组和探针，以及50mM MgCl 2、10×PCR buffer、dNTP/dUTP混合溶液、UDG酶和Taq DNA聚合酶，通过PCR体外扩增法进行病原体检测。其中，引物和探针tpi-F、tpi-R、tpi-P、tcdB-F、tcdB-R、tcdB-P、HP-F、HP-R、HP-P的终浓度比依次为3：3：2：4：4：2.5：2：2：1.5。
阳性对照液由含tpi基因和tcdB基因的质粒制备而成，使用紫外分光光度计对其浓度进行测定，稀释后作为本试剂盒的阳性质控品；阴性对照液为灭菌超纯水。
内标工作液为合成质粒，不含tpi及tcdB基因扩增序列，委托上海捷瑞生物工程有限公司合成内标扩增区域片段(HP)，并插入到pGHn质粒载体上，根据紫外分光光度计测定OD值计算拷贝数，进行梯度稀释后，作为本试剂盒的内标工作液。
实施例2 试剂盒对临床样本的检测
使用实施例1试剂盒检测临床样本中的艰难梭菌，具体步骤如下：
1、样本设置
采集人粪便样本，样本中不应混有尿液、便池水、消毒剂，不应接触便池、卫生纸、尿不湿等，以防样本受到污染或破坏。设置10份艰难梭菌阳性临床样本(P1-P10)和10 份艰难梭菌阴性临床样本(N1-N10)，其中阳性样本包括艰难梭菌产毒菌株(P1-P5)和不产毒菌株(P6-P10)。所有临床粪便样本均经过广州军区总医院检验科的分离培养、质谱鉴定和毒素Sanger测序的验证。
2、粪便基因组DNA的提取
(1)挑取50～200mg粪便于1.5mL离心管中，加入400μL生理盐水溶解，于3000rpm瞬时离心20s；
(2)吸取200μL的上清液至新的2mL离心管中，加入600μL含有内标(内标2μL/人份)的裂解液，涡旋震荡混匀30s，于95～100℃中热裂解10min；
(3)加入600μL无水乙醇，颠倒混匀，于12000rpm离心30s；
(4)将离心柱置入收集套管中，转移900μL上清液至离心柱上，于12000rpm离心1min，弃去收集管中的废液；
(5)加入500μL洗涤缓冲液I(已加入6mL无水乙醇)，于12000rpm离心1min，弃去收集管中的废液；
(6)加入600μL洗涤缓冲液II(已加入14mL无水乙醇)，于12000rpm离心1min，弃去收集管中的废液；
(7)于12000rpm离心2min；
(8)转移离心柱至新的1.5mL离心管上，在纯化柱正中间加入50μL DNA洗脱液，静置2min；
(9)于12000rpm离心1min洗脱DNA。提取的核酸应立即用于后续检测，不能及时检测的应置于-20℃下保存。
从样本中提取基因组DNA，其中加入内标至临床样本中，以参与并监控核酸提取过程。提取的粪便样品中基因组DNA的相关数据(浓度及OD值)如图1所示。由结果可知，提取的基因组DNA纯度均在理想范围内(OD 260/280：1.8～2.0)；因临床样本差异性，DNA浓度在10～350ng/μL范围内不等。
3、阴性对照核酸提取
吸取200μL阴性对照液至新的2mL离心管中，加入600μL含有内标的裂解液，按照粪便基因组DNA的提取的方法进行核酸提取。
4、加样
向待测样本、阴性对照组和阳性对照组中加入PCR反应体系，混匀，经瞬时离心后，按17μL/管分装至PCR反应管中；向分装好的各反应管分别加入处理好的DNA样品3μL、 阴性对照液3μL及阳性对照液3μL。短暂离心使所有试剂集中到反应管底部(不能有气泡)，确定盖好管盖后，立即进行PCR扩增反应。
5、PCR扩增
(1)将PCR反应管放入PCR仪的样品槽中，按对应名称设置阳性对照、阴性对照和未知样品，并设置样品名称、检测靶标名称；
(2)荧光检测通道选择：选择FAM通道检测tpi；选择HEX/VIC通道检测tcdB；选择Cy5通道检测内标。淬灭基团都选None。
(3)设置PCR反应条件
表4 PCR反应条件
Figure PCTCN2018078623-appb-000003
(4)选择反应体系体积为20μL；
(5)设置完毕，保存文件，运行反应程序。
6、结果判断
(1)质量控制
阴性对照：FAM检测通道和HEX检测通道扩增曲线无对数增长期，Cy5检测通道扩增曲线为对数增长期，且Ct值≤36；
阳性对照：FAM检测通道和HEX检测通道扩增曲线有明显对数增长期，且Ct≤24。
以上要求需在同一次试验中同时满足。否则，本次试验无效，需重新进行。
(2)结果判定
表5 艰难梭菌检测结果判定
Figure PCTCN2018078623-appb-000004
(3)临床样本艰难梭菌检测结果如表6所示：
表6 临床样本艰难梭菌检测结果
Figure PCTCN2018078623-appb-000005
注：“-”代表检测结果为阴性，“+”代表检测结果为阳性。
由表6可以看出，实施例1试剂盒对临床样本P1-P10的检测结果，与厌氧培养后质谱鉴定和毒素sanger测序结果相同；而经厌氧培养法鉴定为艰难梭菌阴性的10例样本中，实施例1试剂盒检测出N1-N10为艰难梭菌tpi和tcdB阴性。因此，实施例1试剂盒检测艰难梭菌的结果与预期结果符合，且操作方便快捷，不易受环境影响。
实施例3 试剂盒性能分析
1、特异性分析
(1)交叉反应
采用实施例1所述的试剂盒和实施例2所述的检测方法，选择与艰难梭菌种属相近或引起相似症状的微生物，如小肠结肠炎耶尔森菌、肠毒性大肠杆菌和蜡状芽孢杆菌等21种为待检样本，评价实施例1试剂盒对艰难梭菌相近的引起相似症状的其它病原体检测的符合率，以分析本试剂盒的特异性。待检样本的浓度测OD值后估算，高于1×10 6个菌/mL，结果如表7和图5所示。
实验结果：除了内标有扩增外，tpi和tcdB靶标均没有检测到扩增峰，表示这些样本均没有扩增，与预期结果符合，临床常见的同种属相近的及引起症状相似的其他病原菌不干扰本试剂盒的检测结果，相互之间不存在交叉反应，表明本试剂盒的分析特异性好。
表7 交叉反应实验分析
Figure PCTCN2018078623-appb-000006
注：“-”代表检测结果为阴性。
(2)内源性干扰
采用实施例1所述的试剂盒和实施例2所述的检测方法，取200mg粪便加入200μL艰难梭菌灭活培养物(7.88×10 4个菌/mL)进行检测，另取等量的艰难梭菌灭活培养物为阳性对照，评价含有内源性物质的样本对本试剂盒灵敏度是否存在影响，以分析这些内源性物质的干扰，结果如表8和图6所示。
实验结果表明，在本试剂盒的实验条件下，艰难梭菌灭活培养物加入粪便后，Ct值的变异系数小于5％，样本中可能存在的内源性物质对试剂盒的检测结果无干扰。
表8 内源性物质的干扰试验分析
Figure PCTCN2018078623-appb-000007
(3)外源性干扰
采用实施例1所述的试剂盒和实施例2所述的检测方法，向浓度为7.88×10 5个菌/mL(P1)的艰难梭菌灭活培养物样本中加入以下干扰物质，另以等量的艰难梭菌灭活培养物为阳性对照，评价含有外源性物质的样本对本试剂盒灵敏度是否存在影响，以考察这些外源性物质的干扰，结果如表9和图7所示。
实验结果表明，艰难梭菌培养物加入其它治疗药物后，试剂盒的检测Ct值变异系数均小于5％，说明在本试剂盒的实验条件下，样本中可能存在的外源性干扰物质对试剂盒的检测结果无干扰。
表9 外源性物质干扰实验分析
Figure PCTCN2018078623-appb-000008
2、灵敏度分析
(1)最低检测限的估计
使用已定值的艰难梭菌灭活培养物，稀释至接近试剂盒灵敏度浓度，再进行3倍梯度稀释，稀释浓度分别为7.88×10 2个菌/mL、2.62×10 2个菌/mL和8.75×10 1个菌/mL。然后以实施例1所述的试剂盒和实施例2中所述的试剂盒检测方法，对上述艰难梭菌灭活培养物进行检测。每个浓度各做3个重复，以分析各浓度样本的检出率。本次实验结果统计 如表10所示。
表10 最低检测限的估测
Figure PCTCN2018078623-appb-000009
从上述的实验结果可以看出，确保100％检出的细菌最低浓度介于2.62×10 2个菌/mL和7.88×10 2个菌/mL之间。
(2)最低检测限的确定
以上述估计最低检出限浓度的细菌样本为初始样本，3倍梯度稀释后，使用实施例1所述的试剂盒和实施例2中所述的试剂盒检测方法，对这些样本进行检测。每个浓度每批试剂盒做20个重复，以分析各浓度样本的检出率，95％以上检出的最低浓度就是检测限。实验结果显示，以3.0×10 2个菌/mL浓度的艰难梭菌阳性检出率为100％，如图8所示。由此表明，本试剂盒的最低检测限为3.0×10 2个菌/mL。
对比例1
设置对照组试剂盒A、B和C，选择动物病毒序列作为内标，分别为HCS、HJ和HSa，以合成质粒添加到样本中参与核酸提取过程，分别设计引物、探针，除了内标引物对、内标荧光探针不同，其余组分和实施例1试剂盒一致。由于tpi的扩增效率远比tcdB高，因此只选择tcdB与内标进行双重荧光PCR检测，其余部分与实施例2的检测方法一致。使用实施例1试剂盒和对照组试剂盒A、B、C分别检测临床样本，每个试剂盒设置4个模板浓度梯度。
结果如图9～12所示，由图可知，在双重荧光PCR体系中，HP扩增曲线最好，HSa次之，而HCS和HJ体系中的内标或tcdB的荧光增幅严重受影响。
使用双重荧光中扩增曲线较好的对照组试剂盒C和实施例1试剂盒，采用实施例2的检测方法进行三重荧光PCR检测临床样本，设置4个模板浓度梯度。
结果如图13～14所示，由图可知，在三重荧光PCR体系中，实施例1试剂盒各靶标均能保持良好的扩增效率，不同浓度呈现梯度分布，扩增曲线较均衡，而HSa内标体体系里tpi和tcdB的扩增曲线会受到影响。
对比例2
设置对照组试剂盒D，除了无内标引物对HP-F和HP-R、内标荧光探针HP-P，其余 组分和实施例1试剂盒一致。采用实施例2的检测方法，使用实施例1试剂盒和对照组试剂盒D分别检测艰难梭梭菌标准菌株ATCC9689(该菌株10倍梯度稀释4次)和特异菌株N1～N10，判断标准菌株ATCC9689和特异菌株的tpi和tcdB靶标Ct值是否发生变化，结果如表11所示。由表可知，使用实施例1试剂盒和未添加内标的对照组试剂盒D，标准菌株ATCC9689的Ct值基本相同，几乎没变化，由此说明，内标并未抑制tpi和tcdB靶标的扩增；此外，特异菌株的tpi和tcdB靶标均未能扩增，说明实施例1试剂盒内标的添加并未造成假阳性的结果。
表11 试剂盒内标的添加对靶标扩增的影响分析
Figure PCTCN2018078623-appb-000010
注：“-”代表检测结果为阴性。

Claims (10)

  1. 一组检测艰难梭菌菌属基因及毒素基因的多重荧光PCR引物组和探针，其特征在于，包括tpi扩增引物对tpi-F和tpi-R，tpi荧光探针tpi-P，tcdB扩增引物对tcdB-F和tcdB-R，tcdB荧光探针tcdB-P，内标引物对HP-F和HP-R，内标荧光探针HP-P；核苷酸序列分别依次如SEQ ID NO:1～SEQ ID NO:9所示。
  2. 根据权利要求1所述多重荧光PCR引物组和探针，其特征在于，所述探针的5’末端结合有荧光物质，3’末端结合有淬灭物质；所述荧光物质为FAM、HEX、TET、JOE、VIC、Cy5中的至少一种；所述淬灭物质为TAMRA、BHQ1、BHQ2、BHQ3中的至少一种。
  3. 根据权利要求2所述多重荧光PCR引物组和探针，其特征在于，所述探针的5’末端结合的荧光物质为FAM、HEX、Cy5中的至少一种，3’末端结合的荧光物质为BHQ1。
  4. 权利要求1～3任一所述多重荧光PCR引物组和探针在制备检测艰难梭菌菌属基因及毒素基因的多重荧光PCR试剂或试剂盒方面的应用。
  5. 一种检测艰难梭菌菌属基因及毒素基因的多重荧光PCR试剂盒，其特征在于，该试剂盒含有对tpi基因、tcdB基因和内标进行检测的试剂。
  6. 根据权利要求5所述的试剂盒，其特征在于，包含权利要求1～3任一所述多重荧光PCR引物组和探针，所述引物组和探针tpi-F、tpi-R、tpi-P、tcdB-F、tcdB-R、tcdB-P、HP-F、HP-R、HP-P的终浓度比依次为3：3：2：4：4：2.5：2：2：1.5。
  7. 根据权利要求5所述的试剂盒，其特征在于，所述试剂盒的多重荧光PCR反应条件为：37℃ 5min；95℃ 5min；95℃ 15s，60℃ 38s，45个循环。
  8. 根据权利要求5～7任一项所述的试剂盒，其特征在于，所述试剂盒还包括核酸提取试剂、多重荧光PCR反应所需试剂、内标工作液、阳性对照液和阴性对照液；所述核酸提取试剂包括裂解液、洗涤缓冲液I、洗涤缓冲液II和DNA洗脱液；所述多重荧光PCR反应所需试剂包括MgCl 2、10×PCR buffer、dNTP/dUTP混合溶液、UDG酶和Taq DNA聚合酶；所述内标工作液为不含tpi及tcdB基因扩增序列的HP质粒；所述阳性对照液为含有tpi及tcdB基因扩增序列的克隆质粒；所述阴性对照液为灭菌超纯水。
  9. 根据权利要求8所述的试剂盒，其特征在于，所述裂解液包括SDS、二水柠檬酸三钠、DTT和糖原。
  10. 一种检测艰难梭菌菌属基因及毒素基因的多重荧光PCR方法，其特征在于，包括 以下步骤：
    S1.提取临床样本粪便基因组DNA和阴性对照组DNA，并向裂解液中加入内标工作液；
    S2.向待测样本、阴性对照组和阳性对照组中加入PCR反应体系；
    S3.采用权利要求5至9任一所述试剂盒进行多重荧光PCR反应；
    S4.收集荧光信号，通过测定DNA的Ct值，进行结果分析。
PCT/CN2018/078623 2018-02-02 2018-05-25 一种检测艰难梭菌菌属基因及毒素基因的多重荧光pcr试剂盒和方法 WO2019148590A1 (zh)

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