CN114317786B - Primer probe combination and kit for detecting 14 respiratory tract infection pathogenic bacteria and application of primer probe combination and kit - Google Patents
Primer probe combination and kit for detecting 14 respiratory tract infection pathogenic bacteria and application of primer probe combination and kit Download PDFInfo
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of genetic engineering, in particular to a primer probe combination for detecting 14 respiratory tract infection pathogenic bacteria, a kit and application thereof, wherein the primer probe combination comprises 14 pairs of primers and probes corresponding to the primers; the kit comprises the primer probe combination and is used for detecting/assisting in detecting 14 respiratory tract infection pathogenic bacteria. The sensitivity of the primer probe combination of the invention to the corresponding pathogenic microorganism is higher than that of the existing primer probe combination; the kit can be divided into 4 groups, each group has 4 channels, 14 pathogenic bacteria are detected simultaneously, and the detection method has the advantages of simple operation, high sensitivity, good specificity and short time, and has good clinical application value.
Description
Technical Field
The invention relates to a primer probe combination and application thereof, in particular to a primer probe combination for detecting 14 respiratory tract infection pathogenic bacteria, a kit and application thereof.
Background
Respiratory pathogen infection is one of the most common diseases in clinic, and about 10% of patients in hospitalized patients die from various respiratory infections every year in millions of people worldwide. Each child is infected approximately 6-9 times per year, and adolescents and adults are infected approximately 2-4 times per year.
Respiratory tract infections are classified into upper respiratory tract infections mainly caused by viruses and lower respiratory tract infections mainly caused by bacteria. Lower respiratory tract infections are the most common cause of outpatient visits. World Health Organization (WHO) publishes: of the 5640 deaths worldwide in 2015, more than half of them were caused by 10 causes, with lower respiratory tract infections (319 tens of thousands) listed third, while of the first 10 deaths in low income countries, the lower respiratory tract is first. Lower respiratory tract infections (lower respiratory tract infection, LRTI) are one of the common diseases that threaten human health. Most lower respiratory tract infection is caused by bacterial infection, bacterial infection or secondary infection often causes exacerbation of respiratory tract disease symptoms, if the pathogen type cannot be timely and accurately determined, and the disease optimal treatment time can be delayed by targeted medication, however, the traditional diagnosis and treatment means at present mainly comprise identification of biochemical indexes, pathogen culture and the like, the method operation accuracy is insufficient, the period is long, accurate auxiliary diagnosis is difficult to provide for doctors, and most of medication is still in an empirical medication stage, so that the period of bacteria for producing drug resistance is shortened, and the timely diagnosis of patients is influenced.
Currently, common methods for detecting bacteria clinically include bacterial culture, PCR method, first generation sequencing and second generation sequencing. Isolation culture and smear microscopy are common means of pathogen detection in clinical laboratories. In the detection of lower respiratory tract infections, the determination of pathogenic bacteria by isolated culture is a clinically accepted "gold standard". The separation culture method has long history of determining pathogenic bacteria, has the advantages of simple operation, low cost and the like, and is widely applied clinically. At present, the statistics, investigation and research of pathogenic microorganisms and detection of drug resistance in China are still based on isolated culture, but the whole process generally comprises pathogen culture, identification and antibiotic susceptibility experiments, so that the detection by adopting an isolated culture method takes 2-4 days or even longer.
The smear microscopic examination method is also an important means for lower respiratory tract infection, has low cost, simple and rapid detection method and high specificity, and particularly has important application value in the detection of mycobacterium tuberculosis. However, the detection result obtained by the smear microscopy method is often closely related to the technology, experience and tolerance of operators, the method cannot accurately identify the bacterial species of specific bacteria, and the detection sensitivity is lower than that of the separation culture method.
In clinical practice, the separation culture method and the smear microscopy method are generally combined to improve the diagnosis accuracy and reduce the false negative and the false positive.
The first generation sequencing method has the characteristics of longer reading length and high accuracy, but has the defects of low cost, high flux, long detection time and incapability of detecting bacteria with low copy number. While the second generation sequencing method is superior to the conventional method (isolation culture method and smear microscopy) in diagnosing rare pathogens, the sensitivity of the second generation sequencing method to common pathogens such as mycobacterium tuberculosis, cryptococcus and the like is inferior to that of the conventional method. For these common pathogens, it is often the case that conventional methods do not detect, nor do second generation sequencing; even if these pathogens are detected by the traditional method, the second generation sequencing still cannot detect or only detects a very small amount of specific fragments, so that the accuracy of the detection is difficult to determine, and the detection method is high in cost and long in period.
The PCR detection technique is a polymerase chain reaction that uses three steps of denaturation-annealing-extension to enable the DNA gene to undergo in vitro melting, similar to an in vitro added, replicated format. Along with the development of technology, PCR is widely applied to the field of microorganisms, has the advantages of good repeatability, strong specificity, simple operation, good stability and the like in the detection process of bacteria, gradually becomes a main mode for detecting common bacteria, can detect bacterial distribution and existing positive rate in real time, judges the pathogenic mechanism and epidemiological characteristics of patient infection according to the result, and can play a role in effective prevention and control.
The multiplex PCR technology is a technology in which two or more pairs of primers are added to a PCR reaction system, thereby realizing simultaneous amplification of a plurality of nucleic acid fragments. The real-time fluorescent quantitative PCR technology realizes the quantitative detection function by adding fluorescent markers into a PCR reaction system. The two methods are combined and applied to the detection of pathogenic bacteria, so that not only can a plurality of microorganisms be detected simultaneously, but also the method has the advantages of high sensitivity, high specificity, high accuracy, less pollution and the like.
In view of the above description, it is highly desirable to find a multiplex PCR detection method that has high sensitivity, good specificity, simple experimental operation, and short time consumption, and is capable of simultaneously detecting a plurality of respiratory tract infection pathogens.
Disclosure of Invention
Aiming at the problems, the invention provides a primer probe combination for detecting 14 respiratory tract infection pathogenic bacteria, a kit and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
primer probe combinations for detecting 14 respiratory tract infection pathogenic bacteria and forward primers for detecting klebsiella pneumoniae are shown as SEQ ID NO:1, the reverse primer is shown as SEQ ID NO:2, the probe is shown as SEQ ID NO:3 is shown in the figure;
The forward primer for detecting staphylococcus aureus is shown as SEQ ID NO:4, the reverse primer is shown as SEQ ID NO:5, the probe is shown as SEQ ID NO:6 is shown in the figure;
The forward primer for detecting haemophilus influenzae is shown as SEQ ID NO:7, the reverse primer is shown as SEQ ID NO:8, the probe is shown as SEQ ID NO: shown as 9;
The forward primer for detecting pseudomonas aeruginosa is shown as SEQ ID NO:10, the reverse primer is shown as SEQ ID NO:11, the probe is shown as SEQ ID NO: shown at 12;
The forward primer for detecting Acinetobacter baumannii is shown as SEQ ID NO:13, the reverse primer is shown as SEQ ID NO:14, the probe is shown as SEQ ID NO: 15;
The forward primer for detecting the chlamydia pneumoniae is shown as SEQ ID NO:16, the reverse primer is shown as SEQ ID NO:17, the probe is shown as SEQ ID NO: shown at 18;
The forward primer for detecting streptococcus pneumoniae is shown as SEQ ID NO:19, the reverse primer is shown as SEQ ID NO:20, the probe is shown as SEQ ID NO: 21;
The forward primer for detecting the escherichia coli is shown as SEQ ID NO:22, the reverse primer is shown as SEQ ID NO:23, the probe is shown as SEQ ID NO: shown at 24;
The forward primer for detecting mycoplasma pneumoniae is shown as SEQ ID NO:25, the reverse primer is set forth in SEQ ID NO:26, the probe is shown as SEQ ID NO: shown at 27;
The forward primer for detecting the pseudomonas maltophilia is shown as SEQ ID NO:28, the reverse primer is set forth in SEQ ID NO:29, the probe is shown as SEQ ID NO: shown at 30;
the forward primer for detecting enterobacter cloacae is shown as SEQ ID NO:31, the reverse primer is shown as SEQ ID NO:32, the probe is shown as SEQ ID NO: indicated at 33;
The forward primer for detecting Legionella pneumophila is shown as SEQ ID NO:34, the reverse primer is set forth in SEQ ID NO:35, the probe is shown as SEQ ID NO: shown at 36;
the forward primer for detecting the mycobacterium tuberculosis is shown as SEQ ID NO:37, the reverse primer is set forth in SEQ ID NO:38, the probe is shown as SEQ ID NO: 39;
The forward primer for detecting methicillin-resistant staphylococcus aureus is shown as SEQ ID NO:40, the reverse primer is shown as SEQ ID NO:41, the probe is shown as SEQ ID NO: shown at 42.
A kit comprising the primer probe combination for detecting 14 respiratory tract infection pathogens.
Further, the detection of the kit is divided into 4 groups, and each group comprises 4 channels respectively:
a first group of detection of staphylococcus aureus, pseudomonas aeruginosa, haemophilus influenzae and klebsiella pneumoniae;
a second set of detection of acinetobacter baumannii, streptococcus pneumoniae and chlamydia pneumoniae;
A third group of detection of E.coli, pseudomonas maltophilia, mycoplasma pneumoniae and Enterobacter cloacae;
The fourth group detects methicillin-resistant staphylococcus aureus, legionella pneumophila and mycobacterium tuberculosis.
Further, the reagents in the kit comprise: 4 groups of primer probe combination liquid, TAKRA enzyme Mix and DEPC water.
Further, the concentration of any primer contained in the primer probe combination liquid in any single group of the 4 groups is 5 mu mol/L, and the concentration of any probe is 2.5 mu mol/L.
The application of the kit in detecting/assisting in detecting various respiratory tract infection pathogenic bacteria.
Further, the method is characterized in that the specific steps of the application are that the sample to be detected is taken to extract the nucleic acid of the sample to be detected, then the sample to be detected is respectively mixed with the reagent in the kit to prepare 4 groups of reaction systems, the 4 groups of reaction systems are uniformly mixed in an oscillating way and centrifuged, the PCR amplification reaction is carried out, 4 groups of amplification curve graphs and CT values are obtained, and then the positive/negative of the sample to be detected is judged according to the obtained 4 groups of amplification curve graphs and CT values.
Further, the reaction system is characterized in that the single group of reaction system comprises 4 mu L, TAKRA of nucleic acid of a sample to be detected, 10 mu L of Mix of enzyme and 2 mu L of single group primer probe combination liquid, and DEPC water is added to 20 mu L.
Furthermore, in the application process, positive quality control products and negative quality control products are detected.
Further, the conditions of the PCR amplification reaction were 95℃for 30s,1 cycle, 95℃for 10s, 60℃for annealing extension for 1min, and fluorescence was collected for 40 cycles.
The primer probe combination, the kit and the application thereof for detecting 14 respiratory tract infection pathogenic bacteria have the beneficial effects that:
The sensitivity of the primer probe combination provided by the invention to detection of corresponding pathogenic bacteria is higher than that of the existing primer probe combination;
The kit can be divided into 4 groups, each group comprises 4 channels, 14 respiratory tract infection pathogenic bacteria are detected simultaneously, and the detection method is simple and convenient to operate, high in sensitivity, good in specificity and short in time, and has good clinical application value.
In the kit, the reference genes are added in the second group, whether the sample nucleic acid is successfully extracted or not can be judged through the normal or abnormal condition of the reference genes, for example, if the reference genes in the sample PCR result have no CT value, the problem is solved in the sample acquisition or extraction process, the nucleic acid is not extracted, the sample nucleic acid extraction or the sample re-acquisition is required, and the false negative result is avoided;
the kit can realize multiple detection of samples and can detect the samples in batches.
Drawings
FIG. 1 is a diagram showing the results of a single PCR screening experiment using Kpn primer probe combinations in example 1 of the present invention;
FIG. 2 is a diagram showing the results of a Sau primer probe combination single PCR screening experiment in example 1 of the present invention;
FIG. 3 is a diagram showing the results of a Hin primer probe combination single PCR screening experiment in example 1 of the present invention;
FIG. 4 is a graph showing the results of a Pae primer probe combination single PCR screening experiment in example 1 of the present invention;
FIG. 5 is a diagram showing the results of a screening experiment using the combination of Aba primer probes in example 1 of the present invention;
FIG. 6 is a graph showing the results of a Cpn primer probe combination multiplex PCR screening experiment in example 1 of the present invention;
FIG. 7 is a diagram showing the results of a single PCR screening experiment using the Spn primer probe combination in example 1 of the present invention;
FIG. 8 is a diagram showing the result of a screening experiment of the Eco primer probe combination multiplex PCR in example 1 of the present invention;
FIG. 9 is a diagram showing the results of a screening experiment using a combination of Mpn primer probes in example 1 of the present invention;
FIG. 10 is a diagram showing the result of a single PCR screening experiment of the combination of the Pma primer probe in example 1 of the present invention;
FIG. 11 is a graph showing the results of a screening experiment using Ecl primer probe combinations in example 1 of the present invention;
FIG. 12 is a diagram showing the results of a Len primer probe combination single PCR screening experiment in example 1 of the present invention;
FIG. 13 is a graph showing the results of a screening experiment using Mtb primer probe combinations for single PCR in example 1 of the present invention;
FIG. 14 is a diagram showing the results of a screening experiment of Mrs primer probe combination single PCR in example 1 of the present invention;
FIG. 15 is a graph showing the results of a first mixing screen experiment of a first set of primer probe combinations in example 1 of the present invention;
FIG. 16 is a graph showing the results of a second mixing screen experiment with a first set of primer probes according to example 1 of the present invention;
FIG. 17 is a diagram showing the result of a third mixing screen experiment of the first set of primer probe combinations in example 1 of the present invention;
FIG. 18 is a graph showing the results of a first mixing screening experiment with a second set of primer probe combinations according to example 1 of the present invention;
FIG. 19 is a graph showing the results of a second mixing screen experiment with a second set of primer probes according to example 1 of the present invention;
FIG. 20 is a diagram showing the result of a third mixing screen experiment of the second primer probe combination in example 1 of the present invention;
FIG. 21 is a graph showing the results of a first mixing screen experiment of a third set of primer probe combinations in example 1 of the present invention;
FIG. 22 is a graph showing the results of a second mixing screen experiment with a third set of primer probes according to example 1 of the present invention;
FIG. 23 is a diagram showing the result of a third mixing screen experiment with a third set of primer probes according to example 1 of the present invention;
FIG. 24 is a graph showing the results of a fourth mixing screen experiment with a third set of primer probe combinations according to example 1 of the present invention;
FIG. 25 is a graph showing the results of a first mixing screening experiment with a fourth set of primer probe combinations according to example 1 of the present invention;
FIG. 26 is a diagram showing the result of a second mixing screen experiment with a fourth set of primer probes according to example 1 of the present invention;
FIG. 27 is a diagram showing the result of a third mixing screen experiment with a fourth set of primer probes according to example 1 of the present invention;
FIG. 28 is a graph showing the results of a test for sensitivity of the kit to Sau pathogen in example 3 of the present invention;
FIG. 29 is a graph showing the results of a test for the sensitivity of the kit of example 3 of the present invention to Pae's pathogenic bacteria;
FIG. 30 is a graph showing the results of a test for the sensitivity of the kit to Hin pathogenic bacteria in example 3 of the present invention;
FIG. 31 is a graph showing the results of a test for the sensitivity of the kit to Kpn pathogenic bacteria in example 3 of the present invention;
FIG. 32 is a graph showing the results of a test for the sensitivity of the kit to Aba pathogen in example 3 of the present invention;
FIG. 33 is a graph showing the results of a test for the sensitivity of the kit to Spn pathogenic bacteria in example 3 of the present invention;
FIG. 34 is a graph showing the results of a test for the sensitivity of the kit of example 3 of the present invention to Cpn pathogenic bacteria;
FIG. 35 is a graph showing the result of the sensitivity test of the kit of example 3 of the present invention to Eco pathogenic bacteria;
FIG. 36 is a graph showing the result of the sensitivity test of the kit of example 3 of the present invention to Pma pathogenic bacteria;
FIG. 37 is a graph showing the results of a test for the sensitivity of the kit to Mpn pathogenic bacteria in example 3 of the present invention;
FIG. 38 is a graph showing the results of a test for the sensitivity of the kit to Ecl pathogens in example 3 of the present invention;
FIG. 39 is a graph showing the results of a test for the sensitivity of the kit of example 3 of the present invention to Mrs pathogenic bacteria;
FIG. 40 is a graph showing the results of a test for the sensitivity of the kit to Len's pathogenic bacteria in example 3 of the present invention;
FIG. 41 is a graph showing the results of a test for the sensitivity of the kit to Mtb pathogenic bacteria in example 3 of the present invention.
Detailed Description
The following description of the technical solution in the embodiments of the present invention is clear and complete. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
Example 1 design and screening of primer probe combinations
One) design of primer probe combination
1) Primer probe combination design
Through literature review and market research, 14 target pathogens of respiratory tract and specific genes thereof are determined, and complete genes/partial fragments of the 14 target pathogens are downloaded through NCBI query, so that corresponding DNA sequences of 14 pathogens are obtained, namely klebsiella pneumoniae (Kpn), staphylococcus aureus (Sau), haemophilus influenzae (Hin), pseudomonas aeruginosa (Pae), acinetobacter baumannii (Aba), chlamydia pneumoniae (Cpn), streptococcus pneumoniae (Spn), escherichia coli (Eco), mycoplasma pneumoniae (Mpn), pseudomonas maltophilia (Pma), enterobacter cloacae (Ecl), pneumophilia (Len), mycobacterium tuberculosis (Mtb) and methicillin-resistant staphylococcus aureus (Mrs). The obtained corresponding DNA sequences of 14 target pathogenic bacteria are guided into biological software such as Beacon Designer, oligo, PRIMER PRIMER and the like, a primer probe combination is designed by combining a primer probe design principle and personal probe design experience, the sequence of the designed primer probe combination is input into NCBI for specific comparison, and a single primer probe combination which can amplify the corresponding pathogenic bacteria and does not specifically amplify other pathogenic bacteria is screened out, wherein the specific details are shown in table 1.
TABLE 1 amplification primers and probes for 14 respiratory pathogens (sequence 5'to 3')
The 5 'end of the probe is marked with one of fluorescent report groups FAM, VIC, ROX or CY5, and the 3' end is marked with fluorescent quenching groups BHQ1, BHQ2 and BHQ3.
And respectively combining the designed primer probes and entrusting the synthesis to a general biological company.
2) Primer probe combination screening
21 Construction of a Single PCR System
The PCR amplification experiments were performed separately for the single primer probe combinations in Table 1, and the specific procedure was as follows:
Respectively taking single primer probe combinations in the table 1, and diluting to 10 mu mol/L by adopting DEPC water to obtain single primer probe combination liquid of corresponding pathogenic bacteria (the concentration of a forward primer, a reverse primer and a probe in the single primer probe combination liquid is 10 mu mol/L respectively);
Respectively taking 14 pathogenic bacteria plasmids, diluting the plasmids of each pathogenic bacteria by 10000 times with DEPC water to obtain a plasmid solution of a corresponding single pathogenic bacteria;
And respectively taking 10 mu L of TAKRA enzyme Mix (purchased from TAKRA, product name Premix Ex TapTM (Probe qPCR)) and 4 mu L of DEPC water, sequentially adding the DEPC water into a PCR8 row pipe, adding 2 mu L of single primer Probe combination liquid, and finally adding 4 mu L of corresponding single pathogenic bacteria plasmid solution, mixing (such as adding Kpn pathogenic bacteria plasmid solution into Kpn primer Probe combination liquid), thereby obtaining a corresponding single reaction system. Wherein, the probes of each gene and the amplified plasmid are needed to be corresponding, for example, the probes corresponding to Kpn are needed to be amplified and detected by the plasmid corresponding to Kpn.
The plasmid solution of the corresponding single pathogenic bacteria is replaced by water to prepare a corresponding single control reaction system, wherein the single control reaction system is negative control, and the problem of false positive caused by environmental pollution is judged.
Taking 20 mu L of the single reaction system and the single control reaction system, oscillating, mixing uniformly, centrifuging, respectively transferring into a fluorescent quantitative PCR instrument, performing PCR amplification reaction under the conditions of 95 ℃ pre-denaturation for 30s,1 cycle, 95 ℃ denaturation for 10s, 60 ℃ annealing and extension for 1min, collecting fluorescence, and 40 cycles to obtain the detection results shown in figures 1-14.
The probe quality is screened according to the CT value and the fluorescence intensity, the smaller the CT value is, the better the sensitivity of the probe is, the stronger the fluorescence signal value (namely the higher the Rn value is), and the stronger the binding capacity of the primer probe and the template is. As can be seen from fig. 1 to 14, the three single primer probe combinations in fig. 1 differ by less than 1 CT value, but the amplification curve of the single primer probe combination corresponding to 1b is uniform and stable, so 1b is preferable; in fig. 2, the CT values of the two single primer probe combinations are not different, but the amplification curve of the single primer probe combination corresponding to 2b is better, and the fluorescence value is high and stable, so 2b is preferable; in fig. 3, the two single primer probe combinations have a difference of 1.5 CT values, and 3b is preferred because 3b has a smaller CT value and a higher amplified fluorescence value; in FIG. 4, the CT values of the three single primer probe combinations are not greatly different, and the fluorescence value of the single primer probe combination corresponding to 4b is higher and the stability is best, so 4b is preferable; in fig. 5, the CT values of the two single primer probe combinations are not different, but the fluorescence value of the single primer probe combination corresponding to 5b is higher, so 5b is preferable; in FIG. 6, the indexes of the two groups of single primer probe combinations are not quite different, but the amplification curve of the single primer probe combination corresponding to 6a is slightly better, so 6a is preferable; in fig. 7, three pairs of single primer probe combinations are almost the same as each other in terms of index, so that the three pairs of single primer probe combinations are optional, and then screening is performed when a multiple PRC system is established; in FIG. 8, the CT value of the single primer probe combination corresponding to 8a is poor, the CT values of the single primer probe combinations corresponding to 8b and 8c are not greatly different, but the fluorescence value and the amplification curve of the single primer probe combination corresponding to 8c are obviously better, so that 8c is preferable; in FIG. 9, the CT value of the single primer probe combination corresponding to 9a is smaller, and the amplification curve and fluorescence value are better, so 9a is preferable; in fig. 10, the CT value of the single primer probe set corresponding to 10a is larger, and is directly excluded, and the CT value of the single primer probe set corresponding to 10b is smaller, and the amplification curve is better, so that 10b is preferable; the CT value, amplification curve and fluorescence value of the single primer probe combination corresponding to 11c in FIG. 11 are the best, so 11c is preferred; in FIG. 12, the CT values of the three single-primer probe combinations are almost the same, but the amplification curve and fluorescence value of the single-primer probe combination corresponding to 12a are best, so that 12a is preferable; in FIG. 13, the CT value of the single primer probe combination corresponding to 13b is poor and is directly eliminated, and the amplification curve and fluorescence value of the single primer probe combination corresponding to 13c are good, so that 13c is preferable; in FIG. 14, the CT value, amplification curve and fluorescence value of the single primer probe combination corresponding to 14a are preferably 14a.
22 Establishment of multiple PRC systems
Screening (abbreviated as screening) the single primer probe combination corresponding to 14 pathogenic bacteria screened (abbreviated as screening) in the construction of the single PCR system by utilizing a multiple PRC system.
Considering that the fluorescent quantitative PCR instrument can only collect 4 kinds of fluorescence at the same time, a single group of detection channels can only distinguish 4 kinds of pathogenic bacteria at one time, and the difficulty of carrying out different pathogenic bacteria distinguishing detection by mixing different single primer probe combinations is considered, according to the principle that each group needs to comprise gram negative and gram positive and the other pathogenic bacteria are grouped randomly, the grouping detection is carried out for multiple times, so as to obtain a grouping mode (the grouping detection process needs to be explained, if the mutual interference or the sensitivity detection limit among the primer probe combinations are not at the same level, the primer probe combinations need to be redesigned to be screened, and only the final result is introduced), and the method is specifically as follows:
According to the principle, the grouping is randomly determined, a multiplex PCR amplification experiment is carried out, and the specific multiplex PCR expansion experiment steps are as follows:
Respectively taking corresponding single primer probe combinations according to groups, and diluting with DEPC water until the concentration of any primer in the single groups is 5 mu mol/L and the concentration of any probe is 2.5 mu mol/L to obtain single-group primer probe combination liquid of corresponding groups;
Respectively taking plasmids of 14 pathogenic bacteria, grouping the plasmids of each pathogenic bacteria according to groups, and diluting the plasmids with DEPC water to obtain plasmid solutions of corresponding single groups of pathogenic bacteria;
And respectively taking 10 mu L of TAKRA enzyme Mix and 4 mu L of DEPC water, sequentially adding the water into the PCR8 row tube, adding 2 mu L of single-group primer probe combination liquid, and finally adding 4 mu L of plasmid solution of corresponding single-group pathogenic bacteria, and mixing to obtain a corresponding single-group reaction system.
The plasmid solution of the corresponding single group pathogenic bacteria is replaced by water to prepare a corresponding single group control reaction system, wherein the single group control reaction system is negative control, and the problem of false positive caused by environmental pollution is judged.
Taking 20 mu L of the single-group reaction system and the single-group control reaction system, uniformly mixing by oscillation, centrifuging, respectively transferring to a fluorescent quantitative PCR instrument, carrying out PCR amplification reaction under the conditions of pre-denaturation at 95 ℃ for 30s,1 cycle, denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 1min, collecting fluorescence, and 40 cycles to obtain a detection result.
Firstly, carrying out mixed screening on the corresponding single primer probe combination obtained by the single screen, wherein the mixed screening is qualified and then used, and other single primer probe combinations are selected for carrying out mixed screening again after the mixed screening is unqualified. In the single-screening process, the single-primer probe combination with the 'poor direct elimination of CT value' is removed, the detection results of other single-primer probe combinations are relatively good, and as an alternative, when the preferred single-primer probe combination is unqualified for mixed screening, the single-primer probe combination with other alternative options is selected for mixed screening. Only if these alternatives do not give good results in the mixing screening process, the primer probe combinations are redesigned (it should be noted that only the single primer probe combinations that are finally determined are described in the present invention, and the process of redesigning the primer probe combinations is not described).
The first group was used to detect staphylococcus aureus, pseudomonas aeruginosa, haemophilus influenzae and klebsiella pneumoniae as follows:
the results obtained by selecting the single primer probe combination for the first time in table2 for mixed screening are shown in fig. 15, and it can be seen that the primer probe combination corresponding to klebsiella pneumoniae is optimal in the single screening process, but is detected to be lower in the mixed screening process, so that the grouping is eliminated.
The second single primer probe combination in table 2 was again screened and the results are shown in fig. 16, which shows that klebsiella pneumoniae was not detected, thus excluding the group.
The third single primer probe combination in table 2 was screened and mixed, and the obtained results are shown in fig. 17, and it can be seen that the combination CT value is better, and the mutual interference between the single primer probe combinations is smaller, and the respective priming lines are good, so that the single primer probe combination of the first group is selected. In this regard, the mixing sieve herein only lists a very small portion of the contents and results of the mixing sieve experiment process in order to embody the results.
Table 2 first set of single primer probe combination mixing screening results list
The second group is used for detecting Acinetobacter baumannii, streptococcus pneumoniae and chlamydia pneumoniae, meanwhile, a human genome reference is added in the experimental process, and the human genome reference is a human beta-globin (IC), and the specific detection is as follows:
The results obtained by screening with the single primer probe combinations of Table 3 for the first time are shown in FIG. 18, and it can be seen that Streptococcus pneumoniae is poorly detected and CT values are large, thus excluding the group.
The second single primer probe combination in Table 3 was again mixed and screened to give the results shown in FIG. 19, and it can be seen that the human genome internal reference was not detected, thus excluding the group.
The third single primer probe combination in Table 3 was screened and the results are shown in FIG. 20, which shows that the combination was less than ideal for detection of human genome, but the CT value for Acinetobacter baumannii and Chlamydia pneumoniae was better than the second, and the fluorescence signal of the amplification curve was better than the second. The problem of poor detection limit in the human genome can be improved by adjusting experiments later. So that the group is selected as a single primer probe combination of the second group.
TABLE 3 second set of single primer probe combination mixing screening results list
The third group was used to detect E.coli, pseudomonas maltophilia, mycoplasma pneumoniae and Enterobacter cloacae, and the specific detection was as follows:
the results obtained by mixing and screening with the single primer probe combinations of Table 4 for the first time are shown in FIG. 21, and it can be seen that Enterobacter cloacae was poorly detected, so that the grouping was excluded.
The second single primer probe combination in Table 4 was again screened and the results are shown in FIG. 22, which shows that neither Enterobacter cloacae nor Pseudomonas maltophilia was detected, thus excluding the group.
The third single primer probe combination in Table 4 was screened and the results are shown in FIG. 23, which shows that the amplification curves of Enterobacter cloacae and Pseudomonas maltophilia were poor, thus excluding the group.
The fourth single primer probe combination in table 4 was mixed and screened to obtain the results shown in fig. 24, and it can be seen that the CT value and the amplification curve of the combination are both good, and the mutual infection between the single primer probe combinations is small, and the respective priming lines are good, so that the group is selected as the single primer probe combination of the third group.
Table 4 third set of single primer probe combination mixing screening results list
The fourth group is used for detecting methicillin-resistant staphylococcus aureus, legionella pneumophila and mycobacterium tuberculosis, and the specific detection is as follows:
The results obtained by mixing and screening with the single primer probe combinations of Table 5 for the first time are shown in FIG. 25, and it can be seen that Mycobacterium tuberculosis is not well detected and the amplification curve is not uniform, so that the grouping is excluded.
The second single primer probe combination in Table 5 was again selected for mixing and screening, and the results are shown in FIG. 26, which shows that methicillin-resistant staphylococcus aureus was not detected, thus excluding the group.
The third single primer probe combination in table 5 was mixed and screened to obtain the result shown in fig. 27, and it can be seen that the combination has a small CT value and a better amplification curve, and the single primer probe combinations have small mutual interference and good alignment, so that the single primer probe combination is selected as the fourth single primer probe combination.
Table 5 fourth set of single primer probe combination mixing screening results list
And finally determining the better primer probe combination as follows:
The forward primer for detecting klebsiella pneumoniae is shown as SEQ ID NO:1, the reverse primer is shown as SEQ ID NO:2, the probe is shown as SEQ ID NO:3 is shown in the figure; the forward primer for detecting staphylococcus aureus is shown as SEQ ID NO:4, the reverse primer is shown as SEQ ID NO:5, the probe is shown as SEQ ID NO:6 is shown in the figure; the forward primer for detecting haemophilus influenzae is shown as SEQ ID NO:7, the reverse primer is shown as SEQ ID NO:8, the probe is shown as SEQ ID NO: shown as 9; the forward primer for detecting pseudomonas aeruginosa is shown as SEQ ID NO:10, the reverse primer is shown as SEQ ID NO:11, the probe is shown as SEQ ID NO: shown at 12; the forward primer for detecting Acinetobacter baumannii is shown as SEQ ID NO:13, the reverse primer is shown as SEQ ID NO:14, the probe is shown as SEQ ID NO: 15; the forward primer for detecting the chlamydia pneumoniae is shown as SEQ ID NO:16, the reverse primer is shown as SEQ ID NO:17, the probe is shown as SEQ ID NO: shown at 18; the forward primer for detecting streptococcus pneumoniae is shown as SEQ ID NO:19, the reverse primer is shown as SEQ ID NO:20, the probe is shown as SEQ ID NO: 21; the forward primer for detecting the escherichia coli is shown as SEQ ID NO:22, the reverse primer is shown as SEQ ID NO:23, the probe is shown as SEQ ID NO: shown at 24; the forward primer for detecting mycoplasma pneumoniae is shown as SEQ ID NO:25, the reverse primer is set forth in SEQ ID NO:26, the probe is shown as SEQ ID NO: shown at 27; the forward primer for detecting the pseudomonas maltophilia is shown as SEQ ID NO:28, the reverse primer is set forth in SEQ ID NO:29, the probe is shown as SEQ ID NO: shown at 30; the forward primer for detecting enterobacter cloacae is shown as SEQ ID NO:31, the reverse primer is shown as SEQ ID NO:32, the probe is shown as SEQ ID NO: indicated at 33; the forward primer for detecting Legionella pneumophila is shown as SEQ ID NO:34, the reverse primer is set forth in SEQ ID NO:35, the probe is shown as SEQ ID NO: shown at 36; the forward primer for detecting the mycobacterium tuberculosis is shown as SEQ ID NO:37, the reverse primer is set forth in SEQ ID NO:38, the probe is shown as SEQ ID NO: 39; the forward primer for detecting methicillin-resistant staphylococcus aureus is shown as SEQ ID NO:40, the reverse primer is shown as SEQ ID NO:41, the probe is shown as SEQ ID NO: shown at 42.
The 14 primer probe combinations were divided into the following 4 groups:
a first group of detection of staphylococcus aureus, pseudomonas aeruginosa, haemophilus influenzae and klebsiella pneumoniae;
a second set of detection of acinetobacter baumannii, streptococcus pneumoniae and chlamydia pneumoniae;
A third group of detection of E.coli, pseudomonas maltophilia, mycoplasma pneumoniae and Enterobacter cloacae;
The fourth group detects methicillin-resistant staphylococcus aureus, legionella pneumophila and mycobacterium tuberculosis.
3) Preparation and application of kit
The primer probe combination and grouping preparation kit determined in the establishment of the multiple PRC system is utilized, the prepared kit comprises 4 groups of primer probe combination liquid (wherein the concentration of any primer contained in any single group of primer probe combination liquid is 5 mu mol/L, the concentration of any probe is 2.5 mu mol/L, namely, the concentration of the primer contained in the first group of primer probe combination liquid for detecting staphylococcus aureus, pseudomonas aeruginosa, haemophilus influenzae and klebsiella pneumoniae is 5 mu mol/L, and the concentration of the probe for detecting staphylococcus aureus, pseudomonas aeruginosa, haemophilus influenzae and klebsiella pneumoniae is 2.5 mu mol/L), TAKRA enzyme Mix and DEPC water.
During detection, extracting nucleic acid of a sample to be detected from the sample to be detected; sequentially adding TAKRA enzyme mix and 4 mu L DEPC water into a PCR8 row tube, adding 2 mu L single-group primer probe combination liquid, finally adding 4 mu L sample nucleic acid to be detected to obtain a single-group reaction system (note that a total of 4 groups of reaction systems containing different single-group primer probe combination liquids are required to be prepared for one sample detection), oscillating and uniformly mixing, centrifuging, respectively transferring into a fluorescent quantitative PCR instrument for PCR amplification reaction under the conditions of 95 ℃ presegregation 30S,1 cycle, 95 ℃ denaturation 10S, 60 ℃ annealing extension 1min, collecting fluorescence, 40 cycles, obtaining 4 groups of amplification curve graphs and CT values, respectively analyzing the detection results according to the 4 groups of amplification curve graphs and the CT values, and judging that the fluorescence curve is an S-shaped curve and CT is less than or equal to 37 in FAM, VIC, ROX or CY5 channels, and judging positive; no typical S-shaped amplification or CT is more than 39, and CT of an internal standard is less than or equal to 40, and the internal standard is judged to be negative; if the CT value is between 37 and 39, retesting is carried out, the fluorescence curve is an S-shaped curve, the CT value is more than or equal to 37 and less than or equal to 39, the result is positive, otherwise, the result is negative; and when the judgment result is positive, the sample to be tested contains the pathogenic bacteria.
Meanwhile, in the detection process, positive quality control products and negative quality control products are used for respectively replacing samples to be detected, and detection is carried out according to the method so as to judge whether the problems of false negative or false positive caused by the reasons that the reaction reagent does not reach the standard or environmental pollution and the like exist. And when the problem of false negative or false positive exists, carrying out corresponding pathogen detection on the sample to be detected again.
Example 2 specificity test of primer probe combinations
The implementation adopts 14 pathogenic bacteria positive plasmid standard substances to carry out a cross experiment to verify the specificity, and the specific steps are as follows:
Taking the first group of primer probe combinations as an example, taking a 96-well plate, respectively adding the first group of primer probe combinations into the holes in the same row, namely adding the first group of 4 single primer probe combinations into each tube of the first row, and correspondingly adding the first group of primer probe combinations to the fourth group of primer probe combinations into each tube of the 1 st to 4 th rows, wherein the total number of the primer probe combinations is 4. And then the same single-group pathogenic bacteria positive plasmids are respectively added into the same tube array hole, namely 4 pathogenic bacteria positive plasmids corresponding to the first group of primer probe combinations are respectively added into the first tube array hole, so that the same single-group pathogenic bacteria positive plasmids corresponding to the first group of primer probe combinations to the fourth group of primer probe combinations are respectively and correspondingly added into each tube of the 1 st to 4 th rows, each primer probe and any one of 14 pathogenic bacteria positive plasmids have independent contact opportunities, and each CT value is counted through the cross experiment, so that the specificity of the primer probe combination is verified. In addition, the pathogenic bacteria positive plasmid compositions of helicobacter pylori, moraxella catarrhalis, enterococcus faecium, streptococcus mutans and Staphylococcus epidermidis were added to each tube of the fifth column, and the specificity of the primer probe combination of the present invention was further verified by performing a crossover experiment with the primer probe combination of the present invention, and the specific multiplex PCR amplification experimental method was the same as the multiplex PCR amplification experimental method established by the multiplex PRC system in example 1, and the experimental results are shown in the following table:
TABLE 6 list of results of specific experiments for primer probe combinations
As can be seen from the results of Table 6, the single primer probe combinations of any one of the 4 groupings of the present invention only amplified with their corresponding pathogenic bacteria positive plasmids, while the other pathogenic bacteria positive plasmids were not amplified, demonstrating that the primer probe combinations of the present invention have good specificity.
Example 3 sensitivity experiment of primer probe combinations
The 14 pathogenic bacteria positive plasmids are grouped and mixed according to a pathogenic bacteria grouping mode obtained by establishing a multiplex PCR system in the embodiment 1, ten times of dilutions are respectively carried out, each group is diluted to 10 3～107 copies/mL to obtain 5 pathogenic bacteria positive plasmid solutions (the concentrations are 10 3copies/mL、104copies/mL、105copies/mL、106 copies/mL and 10 7 copies/mL respectively), the preparation and the application of the kit in the embodiment 1 are used for amplifying the 5 pathogenic bacteria positive plasmid solutions in different groups to obtain detection sensitivity corresponding to 14 pathogenic bacteria, the specific results are shown in figures 28-41, and the specific results can be known in the figures, and the standard curve and R 2 values of the 14 pathogenic bacteria detection are shown in the table 7:
TABLE 7 Standard Curve and R 2 values for the detection of the kit of the invention
As can be seen from Table 7, the sensitivity of the kit of the present invention can reach 10 2-103 copies/mL, which is superior to most products.
Example 4 detection Limit stability experiment of primer probe combinations
Grouping fixed value standard substances of 14 pathogenic bacteria positive plasmids according to the embodiment 1, and carrying out gradient dilution to the detection limit concentration (see tables 8-11) to obtain a stability experiment sample;
The preparation and application of the kit in example 1 are used for respectively carrying out 20 times of repeated measurement on the same batch of stability test samples, and the result judgment standard is that at least 18 times of target targets (namely corresponding pathogenic bacteria) must be detected, the detection rate is more than or equal to 90 percent, and the specific results are shown in tables 8-11:
TABLE 8 detection limit stability test results for the first set of kits of the invention
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TABLE 9 stability test results of the second set of kits of the invention
TABLE 10 stability test results of the third group of kits of the invention
TABLE 11 stability test results of the fourth group of kits of the invention
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As can be seen from tables 8-11, the kit of the invention has low detection limit and good stability, and is superior to most products on the market.
Example 5 actual clinical sample detection of primer probe combinations
The clinical samples adopted in the embodiment are from sputum samples collected by hospitals in langfang urban areas (the voluntary principle of collectors), the sputum amount is more than 1mL, 1047 samples are taken, pathogenic bacteria nucleic acid is extracted from the sputum samples, the kit and the detection method are used for detection, and the positive rate of detection is 65.4% by comparison with an internal reference substance and a positive reference substance, and the time is 2 hours.
And detecting the pathogen nucleic acid extracted from the nasopharyngeal swab sample by adopting a traditional pathogen culture method, wherein the positive rate of detection is 65.4%, and the time is 7d.
The detection result of the actual clinical sample of the kit is consistent with the detection result of the traditional pathogenic bacteria culture method, but the time is obviously shorter than that of the traditional pathogenic bacteria culture method.
The specific test results of the actual clinical samples are shown in the following table:
table 12 list of test results for actual clinical samples
| Pathogenic bacteria name abbreviation | The positive cases detected by the kit of the invention | Number of cases of yang diseases detected by traditional method |
| Sau | 170 | 170 |
| Pae | 3 | 3 |
| Hin | 9 | 9 |
| Kpn | 25 | 25 |
| Aba | 5 | 5 |
| Spn | 16 | 16 |
| Cpn | 189 | 189 |
| Eco | 4 | 4 |
| Pma | 52 | 52 |
| Mpn | 2 | 2 |
| Ecl | 173 | 173 |
| Mrs | 2 | 2 |
| Len | 21 | 21 |
| Mtb | 14 | 14 |
It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Sequence listing
<110> langfang NodeChamber laboratory laboratories Inc
<120> Primer probe combination for detecting 14 respiratory tract infection pathogens, kit and application thereof
<130> 2021-12
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Claims (4)
1. A primer probe combination for detecting 14 respiratory tract infection pathogenic bacteria is characterized in that,
The forward primer for detecting klebsiella pneumoniae is shown as SEQ ID NO:1, the reverse primer is shown as SEQ ID NO:2, the probe is shown as SEQ ID NO:3 is shown in the figure;
The forward primer for detecting staphylococcus aureus is shown as SEQ ID NO:4, the reverse primer is shown as SEQ ID NO:5, the probe is shown as SEQ ID NO:6 is shown in the figure;
The forward primer for detecting haemophilus influenzae is shown as SEQ ID NO:7, the reverse primer is shown as SEQ ID NO:8, the probe is shown as SEQ ID NO: shown as 9;
The forward primer for detecting pseudomonas aeruginosa is shown as SEQ ID NO:10, the reverse primer is shown as SEQ ID NO:11, the probe is shown as SEQ ID NO: shown at 12;
The forward primer for detecting Acinetobacter baumannii is shown as SEQ ID NO:13, the reverse primer is shown as SEQ ID NO:14, the probe is shown as SEQ ID NO: 15;
The forward primer for detecting the chlamydia pneumoniae is shown as SEQ ID NO:16, the reverse primer is shown as SEQ ID NO:17, the probe is shown as SEQ ID NO: shown at 18;
The forward primer for detecting streptococcus pneumoniae is shown as SEQ ID NO:19, the reverse primer is shown as SEQ ID NO:20, the probe is shown as SEQ ID NO: 21;
The forward primer for detecting the escherichia coli is shown as SEQ ID NO:22, the reverse primer is shown as SEQ ID NO:23, the probe is shown as SEQ ID NO: shown at 24;
The forward primer for detecting mycoplasma pneumoniae is shown as SEQ ID NO:25, the reverse primer is set forth in SEQ ID NO:26, the probe is shown as SEQ ID NO: shown at 27;
The forward primer for detecting the pseudomonas maltophilia is shown as SEQ ID NO:28, the reverse primer is set forth in SEQ ID NO:29, the probe is shown as SEQ ID NO: shown at 30;
the forward primer for detecting enterobacter cloacae is shown as SEQ ID NO:31, the reverse primer is shown as SEQ ID NO:32, the probe is shown as SEQ ID NO: indicated at 33;
The forward primer for detecting Legionella pneumophila is shown as SEQ ID NO:34, the reverse primer is set forth in SEQ ID NO:35, the probe is shown as SEQ ID NO: shown at 36;
the forward primer for detecting the mycobacterium tuberculosis is shown as SEQ ID NO:37, the reverse primer is set forth in SEQ ID NO:38, the probe is shown as SEQ ID NO: 39;
The forward primer for detecting methicillin-resistant staphylococcus aureus is shown as SEQ ID NO:40, the reverse primer is shown as SEQ ID NO:41, the probe is shown as SEQ ID NO: shown at 42.
2. The kit is characterized by comprising a primer probe combination for detecting 14 respiratory tract infection pathogenic bacteria, and specifically comprises the following components:
The detection of the kit is divided into 4 groups, namely:
A first set of tests for staphylococcus aureus, pseudomonas aeruginosa, haemophilus influenzae and klebsiella pneumoniae, the first set comprising:
The forward primer for detecting staphylococcus aureus is shown as SEQ ID NO:4, the reverse primer is shown as SEQ ID NO:5, the probe is shown as SEQ ID NO:6 is shown in the figure;
The forward primer for detecting pseudomonas aeruginosa is shown as SEQ ID NO:10, reverse primer such as seq id no:11, the probe is shown as SEQ ID NO: shown at 12;
The forward primer for detecting haemophilus influenzae is shown as SEQ ID NO:7, the reverse primer is shown as SEQ ID NO:8, the probe is shown as SEQ ID NO: shown as 9;
the forward primer for detecting klebsiella pneumoniae is shown as SEQ ID NO:1, the reverse primer is shown as SEQ ID NO:2, the probe is shown as SEQ ID NO:3 is shown in the figure;
A second set of tests for acinetobacter baumanii, streptococcus pneumoniae and chlamydia pneumoniae, said second set comprising: the forward primer for detecting Acinetobacter baumannii is shown as SEQ ID NO:13, the reverse primer is shown as SEQ ID NO:14, the probe is shown as SEQ ID NO: 15;
The forward primer for detecting the chlamydia pneumoniae is shown as SEQ ID NO:16, the reverse primer is shown as SEQ ID NO:17, the probe is shown as SEQ ID NO: shown at 18;
The forward primer for detecting streptococcus pneumoniae is shown as SEQ ID NO:19, the reverse primer is shown as SEQ ID NO:20, the probe is shown as SEQ ID NO: 21;
a third set of tests for escherichia coli, pseudomonas maltophilia, mycoplasma pneumoniae and enterobacter cloacae, said third set comprising:
The forward primer for detecting the escherichia coli is shown as SEQ ID NO:22, the reverse primer is shown as SEQ ID NO:23, the probe is shown as SEQ ID NO: shown at 24;
The forward primer for detecting the pseudomonas maltophilia is shown as SEQ ID NO:28, the reverse primer is set forth in SEQ ID NO:29, the probe is shown as SEQ ID NO: shown at 30;
The forward primer for detecting mycoplasma pneumoniae is shown as SEQ ID NO:25, the reverse primer is set forth in SEQ ID NO:26, the probe is shown as SEQ ID NO: shown at 27;
the forward primer for detecting enterobacter cloacae is shown as SEQ ID NO:31, the reverse primer is shown as SEQ ID NO:32, the probe is shown as SEQ ID NO: indicated at 33;
a fourth set of detection of methicillin-resistant staphylococcus aureus, legionella pneumophila, mycobacterium tuberculosis, the fourth set comprising:
The forward primer for detecting methicillin-resistant staphylococcus aureus is shown as SEQ ID NO:40, the reverse primer is shown as SEQ ID NO:41, the probe is shown as SEQ ID NO: 42;
The forward primer for detecting Legionella pneumophila is shown as SEQ ID NO:34, the reverse primer is set forth in SEQ ID NO:35, the probe is shown as SEQ ID NO: shown at 36;
the forward primer for detecting the mycobacterium tuberculosis is shown as SEQ ID NO:37, the reverse primer is set forth in SEQ ID NO:38, the probe is shown as SEQ ID NO: 39.
3. The kit of claim 2, wherein the reagents in the kit comprise: 4 groups of primer probe combination liquid, TAKRA enzyme Mix and DEPC water.
4. The kit according to claim 3, wherein the concentration of any one of the primers contained in the primer probe combination liquid in any one of the 4 sets is 5. Mu. Mol/L, and the concentration of any one of the probes is 2.5. Mu. Mol/L.
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| CN116287356A (en) * | 2023-05-16 | 2023-06-23 | 中国医学科学院北京协和医院 | Primer probe composition and kit for detecting multiple respiratory tract pathogenic bacteria and drug resistance genes |
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| CN107338315A (en) * | 2017-08-15 | 2017-11-10 | 中国人民解放军总医院 | Kit for 15 kinds of pneumonia pathogenic bacteria quick detections |
| CN107937578A (en) * | 2017-12-06 | 2018-04-20 | 西安九安生物技术有限公司 | The primer combination of probe and kit of 15 kinds of respiratory tract infection pathogen joint inspections |
| CN110607379A (en) * | 2019-07-25 | 2019-12-24 | 廊坊诺道中科医学检验实验室有限公司 | Multiplex PCR detection method for simultaneously detecting multiple bacteria and mycoplasma and application thereof |
| CN110894533A (en) * | 2019-11-21 | 2020-03-20 | 北京卓诚惠生生物科技股份有限公司 | Nucleic acid reagent, kit and system for detecting lower respiratory tract infectious bacteria |
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| CN107338315A (en) * | 2017-08-15 | 2017-11-10 | 中国人民解放军总医院 | Kit for 15 kinds of pneumonia pathogenic bacteria quick detections |
| CN107937578A (en) * | 2017-12-06 | 2018-04-20 | 西安九安生物技术有限公司 | The primer combination of probe and kit of 15 kinds of respiratory tract infection pathogen joint inspections |
| CN110607379A (en) * | 2019-07-25 | 2019-12-24 | 廊坊诺道中科医学检验实验室有限公司 | Multiplex PCR detection method for simultaneously detecting multiple bacteria and mycoplasma and application thereof |
| CN110894533A (en) * | 2019-11-21 | 2020-03-20 | 北京卓诚惠生生物科技股份有限公司 | Nucleic acid reagent, kit and system for detecting lower respiratory tract infectious bacteria |
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