CN117802252A - Compositions, kits and methods for simultaneous detection of SFGR, SFTSV and HTNV - Google Patents
Compositions, kits and methods for simultaneous detection of SFGR, SFTSV and HTNV Download PDFInfo
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Abstract
The invention provides a composition, a kit and a method for simultaneously detecting SFGR, SFTSV and HTNV, wherein the composition comprises a primer pair and a probe taking an ompA fragment of Spot fever group rickettsia, an L fragment of new bunyavirus and an L fragment of Hantaan virus as detection targets. The triple real-time fluorescent quantitative PCR detection method provided by the invention can detect three pathogens of SFGR, SFTSV and HTNV simultaneously, has high sensitivity, good specificity and good stability, can realize differential diagnosis in early stage of diseases of patients suffering from fever, hemorrhage or biting, and strives for time for targeted treatment of the patients.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a composition, a kit and a method for simultaneously detecting SFGR, SFTSV and HTNV.
Background
The Spot thermal group Rickettsia (Spotted Fever Group Rickettsiae, SFGR) is a group of obligate intracellular parasitic bacteria belonging to the genus Spot thermal group of Rickettsia, rickettsia order Rickettsia. Tick-borne rickettsiosis caused by SFGR is one of the oldest known vector-borne zoonotics. Hard ticks are the primary vehicle for SFGR, which is also detected in some arthropods such as mosquitoes, fleas, mites, etc. Wild animals and domestic animals are the primary hosts of SFGR, humans as occasional and final hosts are only infected in a few cases. Clinical manifestations of patients with spotted fever are fever, rash, headache, regional lymphadenectasis, etc., and typical symptoms are eschar-like lesions formed at sites where ticks or mites bite, but are usually ignored because the lesions are hidden and painless. The early stage of the disease lacks specific clinical symptoms, patients often delay the disease due to misdiagnosis, and the patients can die when serious according to reported complications including pneumonia, meningoepithymitis, disseminated intravascular coagulation and multiple organ failure. If the patient can be diagnosed quickly and accurately in early stage and the antibacterial drug is given, the patient can be cured, and the death rate is reduced. SFGR is widely distributed worldwide, and with the development of diagnostic techniques, the risk of new and recurrent spot fever group rickettsiae is increasingly of concern, but its global risk distribution and disease burden is unknown.
The fever with thrombocytopenia syndrome virus (severe fever with thrombocytopenia syndrome bunyavirus, SFTSV for short), also called "New bunyavirus" or "Dabieshan virus", SFTSV in 2011 was first isolated and identified in China, belonging to the order bunyaviridae, the family of white fiber viruses, genus Banda, which is a single-stranded negative strand RNA virus whose genome contains 3 RNA circular fragments of large (L), medium (M) and small (S). SFTSV is mainly transmitted by biting of ticks, and the medium ticks are long-angle ticks, microplus, and the like, and furthermore, blood and blood secretions of acute-phase patients and cadavers, and the like are also infectious, and can be infected by direct contact. The disease is mainly clinically manifested by acute fever, thrombocytopenia and leucopenia, along with digestive tract symptoms, nervous symptoms and the like, and serious patients die due to multi-organ failure, and the average early death rate is found to be 12 percent, and even 30 percent in partial areas.
Hantaan virus belongs to the order Hantaan virus, the Hantaan virus family, the genus Hantaan virus, enveloped, segmented, single-stranded negative-strand RNA virus, and the virus group comprises S, M and L three segments, encoding nucleoprotein, glycoprotein, polymerase protein, respectively, and is predominantly clinically manifested as Hantaan viral kidney syndrome Hemorrhagic Fever (HFRS) and Hantaan viral lung syndrome (HPS). In the middle of china, the main epidemic strain of hantavirus is HV004. Infection is characterized by three major clinical manifestations, namely fever, bleeding and kidney injury.
Early clinical manifestations of these pathogens are often not specific and most patients develop systemic symptoms such as fever, headache, fatigue and muscle soreness. Timely and appropriate treatment is particularly important in the early stages of clinical disease where these pathogens infect. This requires targeted rapid identification of patients who are in outdoor activity and have a history of fever in the area to determine specific causative factors. The existing laboratory identification methods of three pathogens mainly depend on traditional separation culture, serological detection, immunohistochemistry, conventional PCR in molecular biology, nested PCR, real-time fluorescence PCR and the like, and the methods have the defects of time consumption, labor consumption, low sensitivity, high cost, single pathogen identification, at most 2 joint tests and the like. Although Crispr-Cas nucleic acid detection has been of great interest for detecting new bunyaviruses, detection can be accomplished within 30 minutes, but sensitivity is not practical with fluorescence PCR and joint detection is difficult. The NGS technology has wide pathogen detection coverage, but is time-consuming and labor-consuming, and the cost is far higher than that of real-time fluorescence PCR.
Disclosure of Invention
In order to solve the problems of the existing laboratory identification methods of three pathogens of spot fever group rickettsia (ompA), new bunyavirus (L fragment) and Hantaan virus (L fragment), such as time consumption, labor consumption, low sensitivity, or high cost, or the identification of single pathogen, at most 2 joint tests, and the like, the invention provides a composition, a kit and a method for simultaneously detecting SFGR, SFTSV and HTNV.
The technical scheme provided by the invention is as follows:
in a first aspect, the present invention provides a composition for real-time fluorescent quantitative PCR detection of spot fever rickettsia, new bunyavirus and hantavirus comprising:
primer pair for spot hotrickettsia:
ompA-F：5’-GCTGSAAATATAAAGAAATCGCTT-3’(SEQ ID NO：1)，
ompA-R：5’-ATTARACCAAAGTTATTGAAAGCTTG-3’(SEQ ID NO：2)；
primer pair for new bunyavirus:
DL-F：5’-GGAAGTGCTTTGTGGTAGGATAA-3’(SEQ ID NO：3)，DL-R：5’-CTRTCGTAGATTTGGTCRTACAGG-3’(SEQ ID NO：4)；
first primer pair of hantavirus:
HL-F1：5’-CCRTATGATTTAACRGARYTAGAGAGC-3’(SEQ ID NO：5)，
HL-R1：5’-CAACCATAYTYTGAGCCTCTCTATC-3’(SEQ ID NO：6)；
second primer pair of hantavirus:
HL-F2：5’-CMAAAGGHMGGGATAATTTGCTTAT-3’(SEQ ID NO：7)，
HL-R2：5’-GCATACGGAATCKTAATTGTAAAG-3’(SEQ ID NO：8)；
spot hotrickettsia probe:
5’-ATGCTCCCAATGGTTCAGATGCAC-3’(SEQ ID NO：9)；
probes for the new bunyavirus:
5’-CGTGGAGAATGGGCTGTCTCTTGG-3’(SEQ ID NO：10)；
first probe of hantavirus:
5’-ACACATTCTCCCCATCCTCGGACAA-3’(SEQ ID NO：11)；
second probe of hantavirus:
5'-TGTAACTGCCATGACCATGCAATCA-3' (SEQ ID NO: 12); wherein Y represents a base C or T, R represents a base A or G, and M represents a base A or C.
Preferably, the probes are Taqman probes.
Preferably, probes of the spot fever rickettsia, the new bunyavirus and the hantavirus are each linked to a different fluorescent reporter group; both probes of hantavirus are linked to the same fluorescent reporter group.
Preferably, probes for the Spot thermal rickettsia, new bunyavirus and Hantaa virus are linked to one of FAM, CY3, CY5, TET, VIC, HEX, respectively.
In a second aspect, the invention provides a real-time fluorescent quantitative PCR detection kit for simultaneously detecting the spot heat rickettsia, the new bunyavirus and the Hantaa virus, wherein the kit contains the composition for detecting the spot heat rickettsia, the new bunyavirus and the Hantaa virus by using the real-time fluorescent quantitative PCR.
Further, the kit further comprises at least one of a nucleic acid extraction reagent, a reverse transcriptase, a DNA polymerase, dUTP, a standard positive template, and a reaction buffer.
Further, the standard positive templates are the sequences respectively comprising SEQ ID NOs: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO:16, and four plasmids of the RNA sequence shown in 16. The plasmids are four types of plasmids respectively inserted with SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO:16, and a PUC57 plasmid of the RNA sequence shown in 16.
Further, the primer pair concentration of the spot heat rickettsia is 250-350 pmol/mL, and the probe concentration is 50-150 pmol/mL; the primer pair concentration of the new bunyavirus is 150-250 pmol/mL, and the probe concentration is 50-150 pmol/mL; the primer pair concentration of the Hantaan virus is 550-650 pmol/mL, and the probe concentration is 50-150 pmol/mL. Preferably, the primer pair concentration of the spot hotrickettsia is 300pmol/mL and the probe concentration is 100pmol/mL; the primer pair concentration of the new bunyavirus is 200pmol/mL, and the probe concentration is 100pmol/mL; the primer pair concentration of Hantaan virus was 600pmol/mL and the probe concentration was 100pmol/mL.
In a third aspect, the present invention provides a real-time fluorescent quantitative PCR detection method for simultaneously detecting spot fever rickettsia, new bunyavirus and Hantaa virus for non-diagnostic purposes, comprising:
1) Extracting total RNA of a sample to be detected;
2) Preparing a PCR reaction system containing the composition of claim 1, adding the extracted total RNA into the PCR reaction system, and performing reverse transcription and PCR amplification reaction;
3) The amplified product was analyzed.
Further, the conditions of the PCR amplification reaction are: reverse transcription is carried out at 50 ℃ for 15min; hot start 95 ℃ for 1-5min; denaturation at 95℃for 10-20s; annealing and extending at 60 ℃ for 20-60s. Preferably, the conditions of the PCR amplification reaction are: reverse transcription is carried out at 50 ℃ for 15min; hot start 95 ℃ for 3min; denaturation at 95℃for 10s; annealing is carried out at 60 ℃ for 30s.
Compared with the prior art, the invention has the following advantages:
the invention establishes a rapid, convenient and accurate multiplex real-time Polymerase Chain Reaction (PCR) method, can detect the spot fever rickettsia, the new bunyavirus and the Hantaan virus simultaneously, can realize differential diagnosis in early stage of diseases of patients suffering from fever, hemorrhage or biting, and strives for time for targeted treatment of the patients.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1: a primer probe concentration optimization chart; wherein a is an amplification curve of different primer probe concentrations of the spot heat rickettsia; b is an amplification curve of the probe concentration of different primers of the new bunyavirus; c is an amplification curve of the Hantaan virus at different primer probe concentrations; d is a heat map of different primer probe concentrations of the spot hotrickettsia; e is a heat map of the probe concentration of different primers of the new bunyavirus; f is a heat map of the Hantaan virus at different primer probe concentrations.
Fig. 2: triple real-time fluorescence quantitative amplification curve and standard curve, wherein the amplification curve sequentially comprises 1X 10 from left to right 6 ～1×10 1 Copy/. Mu.L; wherein a is the amplification curve of the spot heat rickettsia; b is the amplification curve of the new bunyavirus; c is the amplification curve of Hantaa virus; d is a standard curve of the spot heat rickettsia; e is a standard curve of the new bunyavirus; f is the standard curve of Hantaan virus.
Fig. 3: triple real-time fluorescent quantitative PCR specificity experiment results; wherein A represents FAM channel; b represents a VIC channel; c represents CY5 channel, note: the positive control added with plasmid only has amplification curve and Ct value, and the other reaction wells added with hepatitis B virus, hepatitis C virus, EB virus and cytomegalovirus have no amplification curve and CT value, and negative results are presented.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. 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.
The invention provides a composition for detecting spot heat rickettsia, new bunyavirus and Hantavirus by real-time fluorescence quantitative PCR, which comprises the following components:
primer pair for spot hotrickettsia:
ompA-F：5’-GCTGSAAATATAAAGAAATCGCTT-3’，
ompA-R：5’-ATTARACCAAAGTTATTGAAAGCTTG-3’；
primer pair for new bunyavirus:
DL-F：5’-GGAAGTGCTTTGTGGTAGGATAA-3’，
DL-R：5’-CTRTCGTAGATTTGGTCRTACAGG-3’；
first primer pair of hantavirus:
HL-F1：5’-CCRTATGATTTAACRGARYTAGAGAGC-3’，
HL-R1：5’-CAACCATAYTYTGAGCCTCTCTATC-3’；
second primer pair of hantavirus:
HL-F2：5’-CMAAAGGHMGGGATAATTTGCTTAT-3’，
HL-R2：5’-GCATACGGAATCKTAATTGTAAAG-3’；
spot hotrickettsia probe:
5’-ATGCTCCCAATGGTTCAGATGCAC-3’；
probes for the new bunyavirus:
5’-CGTGGAGAATGGGCTGTCTCTTGG-3’；
first probe of hantavirus:
5’-ACACATTCTCCCCATCCTCGGACAA-3’；
second probe of hantavirus:
5’-TGTAACTGCCATGACCATGCAATCA-3’；
wherein Y represents a base C or T, R represents a base A or G, and M represents a base A or C.
The primer pair and the probe are distributed by taking the Spot thermal rickettsia ompA gene sequence (geneID: 928425), the novel bunyavirus L fragment sequence (geneID: 40526338) and the Hantaan virus L fragment sequence (gene ID: 2943080) as detection sites, and the primers have no interference, do not form hairpin structures and dimers, and have good sensitivity and specificity to the detected object.
Preferably, the probes are Taqman probes, the 3 'end of each Taqman probe is connected with a fluorescence quenching group, the 5' end of each Taqman probe is connected with a fluorescence reporting group, and when the probes are complete, fluorescent signals emitted by the fluorescence reporting groups are absorbed by the fluorescence quenching groups and do not display fluorescence; during PCR amplification, taq enzyme is used for enzyme digestion and degradation of the probe, a fluorescent reporter group and a fluorescent quenching group are separated, fluorescence is displayed, and each amplified DNA chain has one fluorescent molecule formed, so that the accumulation of fluorescent signals is completely synchronous with the formation of PCR products.
Preferably, probes of the Spot thermal rickettsia, new bunyavirus and Hantavirus are respectively linked to different fluorescent reporter groups, so that the three pathogens display different fluorescence; the two probes of the Hantaan virus are connected with the same fluorescent reporter group, and the Hantaan viruses of different subtypes are detected by the two probes, but the same fluorescent signal accumulation is carried out, so that the Hantaan viruses are detected uniformly.
Preferably, probes for the Spot thermal rickettsia, new bunyavirus and Hantaa virus are linked to one of FAM, CY3, CY5, TET, VIC, HEX, respectively. Preferably, probes for the Spot thermal rickettsia, new bunyavirus and Hantavirus are ligated FAM, VIC, CY, respectively; further preferably, the probe-linked fluorescent reporter group of the spot thermal rickettsia is FAM and the fluorescent quenching group is BHQ1; the fluorescent reporter group connected with the probe of the new bunyavirus is VIC, and the fluorescent quenching group is BHQ2; the fluorescent reporter group connected with the probes of the Hantaan virus is CY5, and the fluorescent quenching group is BHQ2.
The invention provides a real-time fluorescence quantitative PCR detection kit for simultaneously detecting spot heat rickettsia, new bunyavirus and Hantaa virus, which contains the composition for detecting spot heat rickettsia, new bunyavirus and Hantaa virus by using the real-time fluorescence quantitative PCR detection kit.
Further, the kit further comprises at least one of a nucleic acid extraction reagent, a reverse transcriptase, a DNA polymerase, dUTP, a standard positive template, and a reaction buffer.
Further, the standard positive templates are the sequences respectively comprising SEQ ID NOs: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO:16, and four plasmids of the RNA sequence shown in 16. The plasmids are four types of plasmids respectively inserted with SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO:16, and a PUC57 plasmid of the RNA sequence shown in 16.
Further, the primer pair concentration of the spot heat rickettsia is 250-350 pmol/mL, and the probe concentration is 50-150 pmol/mL; the primer pair concentration of the new bunyavirus is 150-250 pmol/mL, and the probe concentration is 50-150 pmol/mL; the primer pair concentration of the Hantaan virus is 550-650 pmol/mL, and the probe concentration is 50-150 pmol/mL. Preferably, the primer pair concentration of the spot hotrickettsia is 300pmol/mL and the probe concentration is 100pmol/mL; the primer pair concentration of the new bunyavirus is 200pmol/mL, and the probe concentration is 100pmol/mL; the primer pair concentration of Hantaan virus was 600pmol/mL and the probe concentration was 100pmol/mL.
The invention provides a real-time fluorescence quantitative PCR detection method for simultaneously detecting spot fever rickettsia, new bunyavirus and Hantaa virus for non-diagnostic purposes, which comprises the following steps:
1) Extracting total RNA of a sample to be detected;
2) Preparing a PCR reaction system containing the composition of claim 1, adding the extracted total RNA into the PCR reaction system, and performing reverse transcription and PCR amplification reaction;
3) The amplified product was analyzed.
Further, the conditions of the PCR amplification reaction are: reverse transcription is carried out at 50 ℃ for 15min; hot start 95 ℃ for 1-5min; denaturation at 95℃for 10-20s; annealing and extending at 60 ℃ for 20-60s. Preferably, the conditions of the PCR amplification reaction are: reverse transcription is carried out at 50 ℃ for 15min; hot start 95 ℃ for 3min; denaturation at 95℃for 10s; annealing is carried out at 60 ℃ for 30s.
In the present invention, the term "CDS" is an abbreviation for Coding sequence, and refers to a sequence encoding a protein product; the term "NCBI" is an abbreviation for National Center for Biotechnology Information and refers to the national center for biotechnology information.
In the examples below, the experimental materials, reagents and instrumentation used were as follows:
(1) Experimental materials
Nucleic acid extraction reagent: fastPure Viral DNA/RNA Mini Kit Pro;
enzyme: 5X Neoscript Fast RT Premix-UNG (Probe qRT-PCR) cat# (M5244): zhuhai Baozhen Biotechnology Co., ltd;
the primers and the double-labeled Taqman probes were synthesized by general biological company of Chuzhou Anhui.
(2) Apparatus and device
Real-time fluorescent quantitative PCR instrument, gentier 96E/96R full-automatic medical PCR analyzer (Tianlong, sian).
Example 1 design of primers and probes
139 spot hotrickettsia OmpA CDS partial sequences, 1365 new bunyavirus L fragment sequences and 373 Hantavirus L fragment sequences are downloaded from NCBI database. The invention selects the conserved sequence through bioinformatics comparison, and selects two sections of conserved regions to design the primer and the probe due to high variability of the orthohantavirus (Orthohantavirus hantanense). The four selected conserved regions were then cloned into the PUC57 plasmid as standard positive templates for the reaction (see table 8 for sequences of the four selected conserved regions).
The invention takes the Spot thermal rickettsia ompA gene sequence (geneID: 928425), the new bunyavirus L fragment sequence (geneID: 40526338) and the Hantaa virus L fragment sequence (geneID: 2943080) as templates, and utilizes primer design software PrimerPrimer5.0 to respectively design upstream and downstream primers of the Spot thermal rickettsia ompA gene, the new bunyavirus L fragment and the Hantaa virus L fragment, and Taqman probes marked by different luciferins of each gene.
The following analysis was performed on the designed primers: interference between primers, hairpin structure and dimers. And their specificity was initially identified by the BLAST function of NCBI. The primers themselves and the primers with complementary sequences between the primers are eliminated through screening, and the primers with single chains of amplified products capable of forming a secondary structure are selected and remained as shown in the table 1.
TABLE 1
Example 2 determination of optimal reaction conditions for triple fluorescence quantitative PCR
The primers and probes in the table 1 are used for detecting the spot heat rickettsia, the new bunyavirus and the Hantaa virus by adopting a multiplex real-time fluorescence quantitative PCR detection method, and the specific steps are as follows:
(1) DNA/RNA extraction: DNA/RNA was extracted using the Norvezan FastPure Viral DNA/RNA Mini KitPro (cat# RC 323-01) kit.
(2) Establishment of single real-time fluorescent quantitative PCR system
T according to primers and probes m The annealing temperature is set to 60 ℃ and the reaction conditions are as follows: reverse transcription is carried out at 50 ℃ for 15min; hot start 95 ℃ for 3min; denaturation at 95℃for 10s; annealing is carried out at 60 ℃ for 30s.
(3) Establishment and condition optimization of triple real-time fluorescent quantitative PCR
(3.1) establishment of triple real-time fluorescence quantitative PCR System
In order to verify the effectiveness and reliability of each pathogen single-detection real-time fluorescence quantitative PCR system. The invention respectively carries out real-time fluorescence quantitative PCR reaction on each single detection system, and the selection application range is 1 multiplied by 10 6 Copy/. Mu.L to 1X 10 2 A10-fold serial dilution of the standard positive plasmid at copy/. Mu.L was used as template, each concentration was tested 3 times and a negative control was set. And drawing a standard curve according to the optimization result of each single-sample system. The single PCR used 25. Mu.L of a single reaction system:
5μL 5×Neoscript Fast RT Premix Buffer(dUTP)；
1μL 25×Neoscript Fast RTase/UNG Mix；
1.5μL F(10μM)；
1.5μL R(10μM)；
0.5μL TaqMan Probe(10μM)；
2. Mu.L of template DNA; remaining ddH 2 O was made up to 25. Mu.L.
Experiments prove that the results of single detection efficiency of the spot fever rickettsia, the new bunyavirus and the hantavirus primers and probes are shown in table 2:
TABLE 2
The result shows that the primer probes designed by the experiment can meet the preliminary detection requirement (the efficiency is 80-120%, R) 2 Greater than 0.9) which the present invention uses for further optimization.
(3.2) optimization of primer and Probe concentration
RT-qPCR experiments were performed with different concentrations of SFGR, SFTSV and HTNV probe primer combinations, and the annealing temperature was set at 60 ℃. The primers were optimized using a matrix method in the range of 200pmol/mL, 300pmol/mL, 400pmol/mL, 500pmol/mL and 600 pmol/mL; probe concentrations were optimized in the range of 100, 150, 200, 250, 300, 350 and 400 pmol/mL. The amplification curve and heat map results of the optimization procedure are shown in FIG. 1.
The Ct values for different primer and probe concentration gradients for the spot hotrickettsia are shown in table 3:
TABLE 3 Table 3
The Ct value results for the different primer and probe concentration gradients for the new bunyavirus are shown in table 4:
TABLE 4 Table 4
The Ct value results for the different primer and probe concentration gradients for hantavirus are shown in table 5:
TABLE 5
The results show that: the optimal primer concentration of the spot hotrickettsia is 300pmol/mL, and the optimal probe concentration is 100pmol/mL; the optimal primer concentration of the novel bunyavirus is 200pmol/mL, and the optimal probe concentration is 100pmol/mL; the optimal primer concentration for Hantaan virus is 600pmol/mL and the optimal probe concentration is 100pmol/mL.
Example 3 sensitivity detection
A 10-fold dilution of the previously constructed plasmid (range 10 was used 6 -10 1 Copy/. Mu.L) determines the analytical sensitivity and standard curve of the assay. Spot fever rickettsia, new bunyavirus and hantavirus were detected with FAM, VIC and Cy5 fluorescent channels, respectively, all reactions were performed in triplicate, and after the reaction was completed, standard curves were made with the machine's own software Medtl System. The amplification curve and standard curve of the reaction are shown in figure 2.
The invention uses digital PCR to detect 10 3 Copy/. Mu.L, 10 2 Copy/. Mu.L and 10 1 The plasmid copy number of copies/. Mu.L was quantified absolutely. Plasmid copy numbers of the spot hotrickettsia, new bunyavirus and hantavirus are respectively: spot thermarickettsia 3262.99 copies/. Mu.L, 326.4 copies/. Mu.L, 22.17 copies/. Mu.L; 3629.44 copies/. Mu.L, 329.7 copies/. Mu.L, 21.51 copies/. Mu.L of the New bunyavirus; hantavirus 3530.33 copies/. Mu.L, 353.6 copies/. Mu.L, 20.13 copies/. Mu.L.
Then, the invention respectively carries out two-time gradient dilution on about 20 copies/mu L of plasmid to determine the lowest detection limit of the triple real-time fluorescence quantitative PCR, and designs 20 multiple holes. The minimum detection limits of SFGR, SFTSV and HTNV are 1108 copies/mL, 1075 copies/mL and 1006 copies/mL respectively, and the detection rates are all more than 95%.
Example 4 specific assay
In order to determine the specificity of the triple real-time fluorescence quantitative PCR method and eliminate false positive caused by other pathogens, the invention adopts common viral nucleic acid in blood such as hepatitis B virus, hepatitis C virus, EB virus, cytomegalovirus and the like extracted from whole blood or serum as a template, and SFGR, SFTSV and HTNV plasmids as positive controls, and uses an optimized triple real-time fluorescence quantitative PCR method to detect and evaluate the specificity. As shown in FIG. 2, only the positive control shows a typical amplification curve, and other pathogens are not amplified, so that the triple real-time fluorescent quantitative PCR method constructed by the invention has good analysis specificity and can be used for specific detection of SFGR, SFTSV and HTNV. The specificity results are shown in figure 3.
EXAMPLE 5 precision detection
To determine the reproducibility of the triple real-time fluorescent quantitative PCR method, the present invention selects 10 4 Gradient copy/. Mu.L and 10 0 Gradient copies/. Mu.L of standard positive plasmid for each pathogen and equal proportion of mix as template were used for repeat experiments within and between groups of SFGR, SFTSV and HTNV, respectively, on consecutive 5 days, each reaction was repeated 3 times. The Coefficient of Variation (CV) of the Cq value of the sample at each concentration in the experiment was calculated to evaluate the reproducibility thereof. The results are shown in Table 6: the intra-and inter-group Coefficient of Variation (CVs) for each pathogen in the triple combination was less than 5%. The triple real-time fluorescence quantitative PCR method established by the invention has good repeatability and high stability.
TABLE 6
Example 6 clinical sample testing
The triple real-time fluorescent quantitative PCR method and the common PCR detection method obtained by consulting literature data established by the invention are used for respectively carrying out clinical sample detection and positive coincidence rate analysis on 321 samples collected in a laboratory. The judgment standard of the triple real-time fluorescence quantitative PCR method is as follows:
1. the Ct value of the detected sample is smaller than 38, and positive is judged when a typical amplification curve appears;
2. the Ct value of the detected sample is greater than 38, and the detection is needed when a typical amplification curve appears. The rechecking result is judged to be positive when the rechecking result is above, otherwise, the rechecking result is judged to be negative;
3. and when no Ct value exists and no amplification curve exists, the negative judgment is made.
The results are shown in Table 7: SFGR detection rate is 17/321 (5.30%), positive coincidence rate is 100%; SFTSV detection rates are 46/321 (14.33%), and positive coincidence rate is 100%; HTNV detection rate is 32/321 (9.97% respectively), and positive coincidence rate is 97%. Sensitivity, specificity, positive predictive value and negative predictive value results are shown in Table 7.
In conclusion, the triple real-time fluorescence quantitative PCR method established by the invention has better sensitivity and specificity, and has the lowest detection limits of 1108 copies/mL, 1075 copies/mL and 1006 copies/mL of SFGR, SFTSV and HTNV respectively, thus having higher clinical application value.
TABLE 7
Note that: the control method is a sample subjected to nested PCR, single real-time fluorescent PCR or sequencing verification.
TABLE 8
The foregoing is only a specific embodiment of the invention to enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. The composition for detecting the spot fever rickettsia, the new bunyavirus and the hantavirus by real-time fluorescence quantitative PCR is characterized in that: comprising the following steps:
primer pair for spot hotrickettsia:
ompA-F：5’-GCTGSAAATATAAAGAAATCGCTT-3’，
ompA-R：5’-ATTARACCAAAGTTATTGAAAGCTTG-3’；
primer pair for new bunyavirus:
DL-F：5’-GGAAGTGCTTTGTGGTAGGATAA-3’，
DL-R：5’-CTRTCGTAGATTTGGTCRTACAGG-3’；
first primer pair of hantavirus:
HL-F1：5’-CCRTATGATTTAACRGARYTAGAGAGC-3’，
HL-R1：5’-CAACCATAYTYTGAGCCTCTCTATC-3’；
second primer pair of hantavirus:
HL-F2：5’-CMAAAGGHMGGGATAATTTGCTTAT-3’，
HL-R2：5’-GCATACGGAATCKTAATTGTAAAG-3’；
spot hotrickettsia probe:
5’-ATGCTCCCAATGGTTCAGATGCAC-3’；
probes for the new bunyavirus:
5’-CGTGGAGAATGGGCTGTCTCTTGG-3’；
first probe of hantavirus:
5’-ACACATTCTCCCCATCCTCGGACAA-3’；
second probe of hantavirus:
5’-TGTAACTGCCATGACCATGCAATCA-3’；
wherein Y represents a base C or T, R represents a base A or G, and M represents a base A or C.
2. The composition for real-time fluorescent quantitative PCR detection of spot heat rickettsia, new bunyavirus and hantavirus according to claim 1, characterized in that: the probes are Taqman probes.
3. The composition for real-time fluorescent quantitative PCR detection of spot heat rickettsia, new bunyavirus and hantavirus according to claim 2, characterized in that: the probes of the spot fever rickettsia, the new bunyavirus and the Hantaa virus are respectively connected with different fluorescent reporter groups, and the two probes of the Hantaa virus are connected with the same fluorescent reporter groups.
4. A composition for real-time fluorescent quantitative PCR detection of spot heat rickettsia, new bunyavirus and hantavirus according to claim 3, characterized in that: probes for Spot thermal rickettsia, new bunyavirus and Hantaa virus are linked to one of FAM, CY3, CY5, TET, VIC, HEX, respectively.
5. A real-time fluorescent quantitative PCR detection kit for simultaneously detecting spot heat rickettsia, new bunyavirus and hantavirus, characterized in that: the kit contains the composition for detecting spot heat rickettsia, new bunyavirus and hantavirus by real-time fluorescence quantitative PCR according to any one of claims 1-4.
6. The real-time fluorescent quantitative PCR assay kit for simultaneous detection of spot hotrickettsia, new bunyavirus and hantavirus according to claim 5, wherein: the kit further comprises at least one of a nucleic acid extraction reagent, a reverse transcriptase, a DNA polymerase, dUTP, a standard positive template and a reaction buffer.
7. The real-time fluorescent quantitative PCR assay kit for simultaneous detection of spot hotrickettsia, new bunyavirus and hantavirus according to claim 5, wherein: the standard positive templates are the sequences respectively containing SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO:16, and four plasmids of the RNA sequence shown in 16.
8. The real-time fluorescent quantitative PCR assay kit for simultaneous detection of spot hotrickettsia, new bunyavirus and hantavirus according to claim 5, wherein: the primer pair concentration of the spot heat rickettsia is 250-350 pmol/mL, and the probe concentration is 50-150 pmol/mL; the primer pair concentration of the new bunyavirus is 150-250 pmol/mL, and the probe concentration is 50-150 pmol/mL; the primer pair concentration of the Hantaan virus is 550-650 pmol/mL, and the probe concentration is 50-150 pmol/mL.
9. The real-time fluorescence quantitative PCR detection method for simultaneously detecting the spot fever rickettsia, the new bunyavirus and the Hantavirus for non-diagnostic purposes is characterized by comprising the following steps of:
1) Extracting total RNA of a sample to be detected;
2) Preparing a PCR reaction system containing the composition of claim 1, adding the extracted total RNA into the PCR reaction system, and performing reverse transcription and PCR amplification reaction;
3) The amplified product was analyzed.
10. The real-time fluorescent quantitative PCR detection method for simultaneous detection of spot fever rickettsia, new bunyavirus and hantavirus for non-diagnostic purposes according to claim 9, wherein: the conditions for the PCR amplification reaction were: reverse transcription is carried out at 50 ℃ for 15min; hot start 95 ℃ for 1-5min; denaturation at 95℃for 10-20s; annealing and extending at 60 ℃ for 20-60s.
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