CN109797207A - A method of detection four resistance mutation sites of plasmodium falciparum k13 gene - Google Patents
A method of detection four resistance mutation sites of plasmodium falciparum k13 gene Download PDFInfo
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Abstract
The invention discloses a kind of methods for detecting four resistance mutation sites of plasmodium falciparum k13 gene, belong to plasmodium falciparum qinghaosu drug resistant gene detection technique field, this method is that the detection of four resistance mutation sites of plasmodium falciparum k13 gene is realized based on GeXP system；This method is specifically includes the following steps: 1) the design primer group primer sets include a pair of of universal primer and four pairs of specific primers for plasmodium falciparum k13 four resistance mutation sites of gene；2) DNA of plasmodium falciparum is extracted；3) amplification system is established；4) amplified production separates；5) type of mutated gene is determined；Above-mentioned four pairs of specific primers are actually to convert the amplification of multipair peculiar specific primer to the amplification of a pair of of universal primer carrying out PCR amplification, so that the amplification efficiency of each template reaches unanimity, without influencing the amplification efficiency of each peculiar specific primer, to achieve the purpose that really high-throughput detection and identification multiple pathogens.
Description
Technical field
The invention belongs to plasmodium falciparum qinghaosu drug resistant gene detection technique fields, and in particular to a kind of detection malignant malaria
The method of protozoon k13 four resistance mutation sites of gene.
Background technique
In recent years, with Chinese international trade, export of labour services it is increased, overseas input-response relation also increased significantly.
Drug resistance plasmodium falciparum is widely distributed in the whole world, causes serious harm to human health.Detect Introduced cases plasmodium falciparum
Related mutation gene, effectively drug resistance to Introduced cases plasmodium falciparum early detection can be carried out, to control in time
The spread speed of plasmodium falciparum and in time adjustment therapeutic regimen, delay the generation of plasmodium resistance, extend making for antimalarial agent
With the time, have great importance to the work for eliminating plasmodium falciparum.
Plasmodium falciparum generates universal drug resistance to traditional antimalarial in world wide, it has also become global malaria is hindered to disappear
An important factor for except process[1].In recent years, in south east asia, discovery qinghaosu drug resistant falciparum malaria protozoon was also qinghaosu
The use of class drug brings potential threat[2].Current research discovery, plasmodium falciparum Kelch spirochetal protein gene
The mutation in site and plasmodium falciparum are closely related to the generation of qinghaosu resistance on (K13-propeller gene, k13)[3]。
Hereafter Talundzic etc.[4], Tun etc.[5]With Wang etc.[6]The malignant malaria of Thailand, Burma and China-Myanmar border prevalence is found respectively
The multiple sites of K13 gene of protozoon strain mutate.Feng etc.[7]The malignant malaria of returned personnel of overseas working to Guangxi infection is former
Worm the study found that C580Y mutation rate highest (2.7%) in all mutational sites of K13 gene.When the certain site hairs of K13 gene
Raw mutation, can weaken the interaction of K13-propeller albumen and receptor protein, lead to the generation of qinghaosu drug resistance[3]Cause
This, with detect K13 gene kelch structural domain it is polymorphic come predict plasmodium falciparum qinghaosu drug resistance have theory support.
Multiplex PCR and multiple fluorescence PCR are two kinds and apply more detection and identification technology in a variety of cause of disease mixed infections.
Multiplex PCR needs multipair primer combination to combine amplification to competition simultaneously in a reaction system, can generate interference between primer, often
Increase pair of primers, the probability for forming complicated primer dimer greatly increases, and sensibility is lower, and multiplex PCR produces in addition more
Object need to be by agarose electrophoresis ability observable as a result, it is difficult to distinguish the band less than 100bp；And multiple fluorescence PCR is due to probe
Mark the fluorophor of different wave length, such as mark it is more, can mutually generate interference it is bigger, sensibility is lower.Multiplex PCR and
The target gene limited amount of multiple fluorescence PCR detection can only generally carry out the detection of 2 to 4 weights, real high pass is all not achieved
Measure the purpose of quick detection and analysis multiple pathogens.
Summary of the invention
The technical problems to be solved by the invention are as follows: multiplex PCR and the target gene quantity of multiple fluorescence PCR detection have
Limit can only generally carry out the detection of 2 to 4 weights, and the mesh of really high-throughput quickly detection and analysis multiple pathogens is all not achieved
's.The present invention provides a kind of method for detecting four resistance mutation sites of plasmodium falciparum k13 gene.
Technical scheme is as follows: a method of detection four resistance mutation sites of plasmodium falciparum k13 gene,
This method is that the detection of four resistance mutation sites of plasmodium falciparum k13 gene is realized based on GeXP system；Plasmodium falciparum k13
Four resistance mutation sites of gene are respectively as follows: Y493H, R539T, I543T and C580Y；
GeXP system combines multiple PCR technique and capillary electrophoresis technique；
This method specifically includes the following steps:
1) design primer group
The primer sets include a pair of of universal primer and for four pairs of plasmodium falciparum k13 four resistance mutation sites of gene
Specific primer, four pairs of specific primers are respectively as follows: Y493H specific primer, R539T specific primer, I543T specificity and draw
Object and C580Y specific primer；
Y493H specific primer is the end connection universal primer sequence composition in Y493H；R539T specific primer is
It is formed in the end connection universal primer sequence of R539T；I543T specific primer is the end connection universal primer in I543T
Sequence composition；C580Y specific primer is the end connection universal primer sequence composition in C580Y；
2) DNA of plasmodium falciparum is extracted
The DNA that plasmodium falciparum is extracted from the blood of human body needs that RNA first is added in the blood of human body before extracting
Enzyme；And the concentration of plasmodium falciparum is not less than 5 × 10 in the blood of human body of detection3copy/μL；
3) amplification system is established
Initiation polyad is combined by four pairs of specific primers described in step 1) and using the universal primer of fluorescent marker
System's amplification, specifically are as follows: by four pairs of specific primers described in step 1), using the universal primer and step 2) of fluorescent marker
In extracted malignant malaria original DNA mix to establish amplification system；
4) amplified production separates
Obtained product after step 3) amplification is subjected to capillary electrophoresis technique separation,
5) type of mutated gene is determined
The PCR product containing fluorescent marker after the completion of amplification in step 4) is detected and calculated through GeXP detection window
The size of amplified fragments compares the size for the amplified fragments that the size of amplified fragments obtains after quality-control product amplification to really
Determine the type of mutated gene.
The working principle of the invention or have the beneficial effect that PCR reaction at the beginning of, first by reversed specific primer and original mRN
In conjunction with carrying out reverse transcription reaction, then synthesize by positive specific primer the second chain of cDNA, hereafter, by forward primer with reversely draw
The specific sequence of object starts PCR reaction by template of cDNA, amplifies the complementary series of universal primer respectively；Again by reactant
Prevailing fluorescent marker universal primer in system is complementary sequence combination, causes following amplification.
It is combined using fluorescent marker universal primer and specific primer and causes multiple system amplification, thus multipair primer
Amplification be converted into the amplification of a pair of of universal primer, so that the amplification efficiency of each template is reached unanimity, the expansion without influencing each primer
Increasing Efficiency, to achieve the purpose that really high-throughput detection and identification multiple pathogens.
The amplification preference of traditional multiple PCR technique caused by this method has been well solved due to PCR selection and PCR drift
Multiplex amplification product be separated into single-stranded by property, this method, detects amplified fragments using capillary electrophoresis separation, Neng Gouqu
Slitting band is less than the amplified production of 100bp.
It further limits, the forward primer sequence of the Y493H specific primer are as follows: AGGTGACACTATAGAATATGG
AAGTGCTGTATTGAATAATTTCTTACAC,
The reverse primer sequences of the Y493H specific primer are as follows: GTACGACTCACTATAGGGAATTTGACGTAACA
CCACAATTATTTCTTCTAG；
The forward primer sequence of the R539T specific primer are as follows: AGGTGACACTATAGAATATAATTGTGGTGTTA
CGTCAAATGGTACA；
The reverse primer sequences of the R539T specific primer are as follows: GTACGACTCACTATAGGGATACATTCGGTATA
ATAGAAGAGCCATCATA；
The forward primer sequence of the I543T specific primer are as follows: AGGTGACACTATAGAATATGGTGTTACGTCAA
ATGGTAGAATTTATTGTACT；
The reverse primer sequences of the I543T specific primer are as follows: GTACGACTCACTATAGGGACTCTCACCATTAG
TTCCACCAATGAC；
The forward primer sequence of the C580Y specific primer are as follows: AGGTGACACTATAGAATAATACCCCTAGATCA
TCAGCTATGTGT；
The reverse primer sequences of the C580Y specific primer are as follows: GTACGACTCACTATAGGGAATTATATAAGAAT
CTGACAATGTGGCAGCT。
It further limits, the universal primer includes upstream primer and downstream primer, the primer sequence of upstream primer are as follows:
AGGTGACACTATAGAATA, the primer sequence of downstream primer are as follows: GTACGACTCACTATAGGGA.
It further limits, the specific amplified clip size of the Y493H is 200bp；The specific amplified segment of the R539T
Size is 113bp；The specific amplified clip size of the I543T is 232bp；The specific amplified clip size of the C580Y is
374bp。
It further limits, is to calculate amplified fragments according to detection segment and standard molecule segment transit time in step 4)
Length.
Detailed description of the invention
Fig. 1 is the PCR reaction product electrophoretogram of R539T specific primer；
Fig. 2 is the PCR reaction product electrophoretogram of I543T specific primer；
Fig. 3 is the PCR reaction product electrophoretogram of C580Y specific primer；
Fig. 4 is the PCR reaction product electrophoretogram of Y493H specific primer；
Fig. 5 is the PCR reaction product electrophoretogram of four pairs of specific primers；
Fig. 6 is the GeXP substance PCR testing result of R539T specific primer；
Fig. 7 is the GeXP substance PCR testing result of Y493H specific primer；
Fig. 8 is the GeXP substance PCR testing result of I543T specific primer；
Fig. 9 is the GeXP substance PCR testing result of C580Y specific primer；
Figure 10 is the GeXP multiplex PCR testing result of the mixture of C580Y specific primer and R539T specific primer；
Figure 11 is the GeXP multiplex PCR testing result of the mixture of R539T specific primer and Y493H specific primer；
Figure 12 is the GeXP multiplex PCR testing result of the mixture of R539T specific primer and I543T specific primer；
Figure 13 is the mixture of C580Y specific primer, R539T specific primer and Y493H specific primer
GeXP multiplex PCR testing result；
Figure 14 is the mixture of I543T specific primer, R539T specific primer and Y493H specific primer
GeXP multiplex PCR testing result；
Figure 15 is I543T specific primer, R539T specific primer, Y493H specific primer and C580Y specificity
The GeXP multiplex PCR testing result of the mixture of primer.
Specific embodiment
It is illustrated below using specific embodiment.Embodiments of the present invention include but is not limited to the following example.
Embodiment 1
1) design primer group
Bibliography and ncbi database, search for the nearly 8 years plasmodium falciparums molecular labeling of resistance to qinghaosu k13 gene order and
Its four SNP sites, and albumen and nucleotide sequence are downloaded from GenBank database, it is carried out using Clustalx software more
Sequence homology compares analysis, by comparison result input GeXP eXpress Profiler tool design four to specific primer.
Four pairs of specific primers are analyzed using 6.0 software of NCBIPrimer-Blast, Primer Premier 5.0 and Oligo
Screening.Plasmodium vivax (Plasmodium vivax), the malariae (Plamodiummalariae) of nearly edge are downloaded simultaneously
Series carries out homologous interference assessment.
Sample collection and plasmodium falciparum DNA are extracted
Positive plasmid quality-control product and positive clinical sample with the configuration of 4 kinds of plasmodium falciparum drug resistance gene mutational sites
This DNA, negative quality-control product is configured as control using the wild type plasmodium plasmid for not carrying drug resistance gene mutated gene.It is positive
Plasmid quality-control product is that the plasmid of synthesis Plasmodium falciparum sweet wormwood plain gene k13 is configured according to a certain concentration.
2) the plasmodium falciparum molecular labeling of resistance to qinghaosu k13 gene information
PF3D7_1343700kelch protein K13[Plasmodium falciparum 3D7]
Gene symbol PF3D7_1343700
Gene description kelch protein K13
Locus tag PF3D7_1343700
Gene type protein coding
RNA name kelch protein K13
Lineage Eukaryota；Alveolata；Apicomplexa；Aconoidasida；Haemosporida；
Plasmodiidae；Plasmodium；Plasmodium(Laverania).
3) the plasmodium falciparum molecular labeling of resistance to qinghaosu k13 gene amino acid sequence
MEGEKVKTKANSISNFSMTYDRESGGNSNSDDKSGSSSENDSNS
FMNLTSDKNEKTENNSFLLNNSSYGNVKDSLLESIDMSVLDSNFDSKKDFLPSNLSRT
FNNMSKDNIGNKYLNKLLNKKKDTITNENNNINHNNNNNNLTANNITNNLINNNMNSP
SIMNTNKKENFLDAANLINDDSGLNNLKKFSTVNNVNDTYEKKIIETELSDASDFENM
VGDLRITFINWLKKTQMNFIREKDKLFKDKKELEMERVRLYKELENRKNIEEQKLHDE
RKKLDIDISNGYKQIKKEKEEHRKRFDEERLRFLQEIDKIKLVLYLEKEKYYQEYKNF
ENDKKKIVDANIATETMIDINVGGAIFETSRHTLTQQKDSFIEKLLSGRHHVTRDKQG
RIFLDRDSELFRIILNFLRNPLTIPIPKDLSESEALLKEAEFYGIKFLPFPLVFCIGG
FDGVEYLNSMELLDISQQCWRMCTPMSTKKAYFGSAVLNNFLYVFGGNNYDYKALFET
EVYDRLRDVWYVSSNLNIPRRNNCGVTSNGRIYCIGGYDGSSIIPNVEAYDHRMKAWV
EVAPLNTPRSSAMCVAFDNKIYVIGGTNGERLNSIEVYEEKMNKWEQFPYALLEARSS
GAAFNYLNQIYVVGGIDNEHNILDSVEQYQPFNKRWQFLNGVPEKKMNFGAATLSDSY
IITGGENGEVLNSCHFFSPDTNEWQLGPSLLVPRFGHSVLIANI
4) the plasmodium falciparum molecular labeling of resistance to qinghaosu k13 gene DNA sequence
ATGGAAGGAGAAAAAGTAAAAACAAAAGCAAATAGTATCTCGAATTTTTCTATGACGTATGATAGGGAA
TCTGGTGGTAACAGCAATAGTGATGATAAAAGCGGAAGTAGTAGCGAGAATGATTCTAATTCATTTATGAATCTAAC
TAGTGATAAAAATGAGAAAACGGAAAATAATAGTTTCCTTTTAAATAATAGTAGTTATGGAAATGTTAAAGATAGCC
TATTAGAATCCATTGATATGAGTGTATTAGATTCGAACTTTGATAGTAAAAAAGATTTTTTACCAAGTAATTTATCA
AGAACATTTAATAATATGTCTAAAGATAATATAGGAAATAAATATTTAAATAAATTGTTAAATAAAAAAAAAGATAC
TATTACAAATGAAAATAATAATATTAATCATAATAATAATAATAATAATCTGACAGCAAATAATATAACTAATAATC
TTATTAATAATAATATGAATTCTCCATCAATTATGAATACCAACAAAAAAGAGAATTTTTTAGATGCAGCAAATCTT
ATAAATGATGATTCTGGATTAAACAATTTAAAAAAATTTTCAACTGTAAATAATGTAAATGATACTTATGAAAAGAA
AATTATTGAAACGGAATTAAGTGATGCTAGTGATTTTGAAAATATGGTAGGTGATTTAAGAATTACATTTATTAATT
GGTTAAAAAAGACACAAATGAATTTTATTCGAGAAAAAGATAAATTATTTAAAGATAAGAAAGAACTAGAAATGGAA
AGAGTACGATTGTACAAAGAATTAGAAAACCGTAAAAATATTGAAGAACAGAAATTACATGATGAAAGAAAGAAATT
AGATATTGATATATCTAATGGTTATAAACAAATAAAAAAAGAAAAAGAAGAACATAGGAAACGATTTGATGAAGAAA
GATTAAGATTTTTACAAGAAATCGATAAAATTAAATTAGTATTATATTTAGAAAAAGAAAAATATTATCAAGAATAT
AAAAATTTTGAGAATGATAAAAAAAAAATTGTTGATGCAAATATTGCTACTGAAACTATGATTGATATTAATGTTGG
TGGAGCTATTTTTGAAACATCTAGACATACCTTAACACAACAAAAAGATTCATTTATAGAGAAATTATTAAGTGGAA
GACATCATGTAACCAGAGATAAACAAGGAAGAATATTCTTAGATAGGGATAGTGAGTTATTTAGAATTATACTTAAC
TTCTTAAGAAATCCGTTAACTATACCCATACCAAAAGATTTAAGTGAAAGTGAAGCCTTGTTGAAAGAAGCAGAATT
TTATGGTATTAAATTTTTACCATTCCCATTAGTATTTTGTATAGGTGGATTTGATGGTGTAGAATATTTAAATTCGA
TGGAATTATTAGATATTAGTCAACAATGCTGGCGTATGTGTACACCTATGTCTACCAAAAAAGCTTATTTTGGAAGT
GCTGTATTGAATAATTTCTTATACGTTTTTGGTGGTAATAACTATGATTATAAGGCTTTATTTGAAACTGAGGTGTA
TGATCGTTTAAGAGATGTATGGTATGTTTCAAGTAATTTAAATATACCTAGAAGAAATAATTGTGGTGTTACGTCAA
ATGGTAGAATTTATTGTATTGGGGGATATGATGGCTCTTCTATTATACCGAATGTAGAAGCATATGATCATCGTATG
AAAGCATGGGTAGAGGTGGCACCTTTGAATACCCCTAGATCATCAGCTATGTGTGTTGCTTTTGATAATAAAATTTA
TGTCATTGGTGGAACTAATGGTGAGAGATTAAATTCTATTGAAGTATATGAAGAAAAAATGAATAAATGGGAACAAT
TTCCATATGCCTTATTAGAAGCTAGAAGTTCAGGAGCAGCTTTTAATTACCTTAATCAAATATATGTTGTTGGAGGT
ATTGATAATGAACATAACATATTAGATTCCGTTGAACAATATCAACCATTTAATAAAAGATGGCAATTTCTAAATGG
TGTACCAGAGAAAAAAATGAATTTTGGAGCTGCCACATTGTCAGATTCTTATATAATTACAGGAGGAGAAAATGGCG
AAGTTCTAAATTCATGTCATTTCTTTTCACCAGATACAAATGAATGGCAGCTTGGCCCATCTTTATTAGTTCCCAGA
TTTGGTCACTCCGTTTTAATAGCAAATATATAA
5) the plasmodium falciparum molecular labeling of resistance to qinghaosu k13 gene mutation site information
ATGGAAGGAGAAAAAGTAAAAACAAAAGCAAATAGTATCTCGAATTTTTCTATGACGTATGATAGGGA
ATCTGGTGGTAACAGCAATAGTGATGATAAAAGCGGAAGTAGTAGCGAGAATGATTCTAATTCATTTATGAATCTA
ACTAGTGATAAAAATGAGAAAACGGAAAATAATAGTTTCCTTTTAAATAATAGTAGTTATGGAAATGTTAAAGATA
GCCTATTAGAATCCATTGATATGAGTGTATTAGATTCGAACTTTGATAGTAAAAAAGATTTTTTACCAAGTAATTT
ATCAAGAACATTTAATAATATGTCTAAAGATAATATAGGAAATAAATATTTAAATAAATTGTTAAATAAAAAAAAA
GATACTATTACAAATGAAAATAATAATATTAATCATAATAATAATAATAATAATCTGACAGCAAATAATATAACTA
ATAATCTTATTAATAATAATATGAATTCTCCATCAATTATGAATACCAACAAAAAAGAGAATTTTTTAGATGCAGC
AAATCTTATAAATGATGATTCTGGATTAAACAATTTAAAAAAATTTTCAACTGTAAATAATGTAAATGATACTTAT
GAAAAGAAAATTATTGAAACGGAATTAAGTGATGCTAGTGATTTTGAAAATATGGTAGGTGATTTAAGAATTACAT
TTATTAATTGGTTAAAAAAGACACAAATGAATTTTATTCGAGAAAAAGATAAATTATTTAAAGATAAGAAAGAACT
AGAAATGGAAAGAGTACGATTGTACAAAGAATTAGAAAACCGTAAAAATATTGAAGAACAGAAATTACATGATGAA
AGAAAGAAATTAGATATTGATATATCTAATGGTTATAAACAAATAAAAAAAGAAAAAGAAGAACATAGGAAACGAT
TTGATGAAGAAAGATTAAGATTTTTACAAGAAATCGATAAAATTAAATTAGTATTATATTTAGAAAAAGAAAAATA
TTATCAAGAATATAAAAATTTTGAGAATGATAAAAAAAAAATTGTTGATGCAAATATTGCTACTGAAACTATGATT
GATATTAATGTTGGTGGAGCTATTTTTGAAACATCTAGACATACCTTAACACAACAAAAAGATTCATTTATAGAGA
AATTATTAAGTGGAAGACATCATGTAACCAGAGATAAACAAGGAAGAATATTCTTAGATAGGGATAGTGAGTTATT
TAGAATTATACTTAACTTCTTAAGAAATCCGTTAACTATACCCATACCAAAAGATTTAAGTGAAAGTGAAGCCTTG
TTGAAAGAAGCAGAATTTTATGGTATTAAATTTTTACCATTCCCATTAGTATTTTGTATAGGTGGATTTGATGGTG
TAGAATATTTAAATTCGATGGAATTATTAGATATTAGTCAACAATGCTGGCGTATGTGTACACCTATGTCTACCAA
AAAAGCTTATTTTGGAAGTGCTGTATTGAATAATTTCTTATAC(Y493H, TAC > CAC) GTTTTTGGTGGTAATAA
CTATGATTATAAGGCTTTATTTGAAACTGAGGTGTATGATCGTTTAAGAGATGTATGGTATGTTTCAAGTAATTTA
AATATACCTAGAAGAAATAATTGTGGTGTTACGTCAAATGGTAGA(R539T, AGA > ACA) ATTTATTGTATT
(I543T, ATT > ACT) GGGGGATATGATGGCTCTTCTATTATACCGAATGTAGAAGCATATGATCATCGTAT GAAA
GCATGGGTAGAGGTGGCACCTTTGAATACCCCTAGATCATCAGCTATGTGT(C580Y, TGT > TAT) GTTGCTTTT
GATAATAAAATTTATGTCATTGGTGGAACTAATGGTGAGAGATTAAATTCTATTGAAGTATATGAAGAAAAAATGAA
TAAATGGGAACAATTTCCATATGCCTTATTAGAAGCTAGAAGTTCAGGAGCAGCTTTTAATTACCTTAATCAAATAT
ATGTTGTTGGAGGTATTGATAATGAACATAACATATTAGATTCCGTTGAACAATATCAACCATTTAATAAAAGATGG
CAATTTCTAAATGGTGTACCAGAGAAAAAAATGAATTTTGGAGCTGCCACATTGTCAGATTCTTATATAATTACAGG
AGGAGAAAATGGCGAAGTTCTAAATTCATGTCATTTCTTTTCACCAGATACAAATGAATGGCAGCTTGGCCCATCTT
TATTAGTTCCCAGATTTGGTCACTCCGTTTTAATAGCAAATATATAA
6) universal primer
Upstream primer: AGGTGACACTATAGAATA
Downstream primer: GTACGACTCACTATAGGGA
7) specific primer
Design four pairs of specific primers primer sequence and four resistance mutation sites specific amplified clip size such as
Shown in table 1；
1 specific primer sequence table of table
8) prepared by standard items
Four SNP sites of the plasmodium falciparum molecular labeling of resistance to qinghaosu k13 gene are downloaded from GenBank database
The mutant sequences of (Y493H, R539T, I543T and C580Y), choose the sequence of specific amplified, and stored DNA concentration is 5 ×
106copy/mL。
Wherein, the PCR amplification sequence and primer information of four pairs of specific primers are as follows:
Y493H, total 603bp
TGGAAGTGCTGTATTGAATAATTTCTTACACGTTTTTGGTGGTAATAACTATGATTATAAGGCTTTATT
TGAAACTGAGGTGTATGATCGTTTAAGAGATGTATGGTATGTTTCAAGTAATTTAAATATACCTAGAAGAAATAATT
GTGGTGTTACGTCAAATGGTAGAATTTATTGTATTGGGGGATATGATGGCTCTTCTATTATACCGAATGTAGAAGCA
TATGATCATCGTATGAAAGCATGGGTAGAGGTGGCACCTTTGAATACCCCTAGATCATCAGCTATGTGTGTTGCTTT
TGATAATAAAATTTATGTCATTGGTGGAACTAATGGTGAGAGATTAAATTCTATTGAAGTATATGAAGAAAAAATGA
ATAAATGGGAACAATTTCCATATGCCTTATTAGAAGCTAGAAGTTCAGGAGCAGCTTTTAATTACCTTAATCAAATA
TATGTTGTTGGAGGTATTGATAATGAACATAACATATTAGATTCCGTTGAACAATATCAACCATTTAATAAAAGATG
GCAATTTCTAAATGGTGTACCAGAGAAAAAAATGAATTTTGGAGCTGCCACATTGTCAGATTCTTATATAAT
R539T, total 603bp
TGGAAGTGCTGTATTGAATAATTTCTTATACGTTTTTGGTGGTAATAACTATGATTATAAGGCTTTATT
TGAAACTGAGGTGTATGATCGTTTAAGAGATGTATGGTATGTTTCAAGTAATTTAAATATACCTAGAAGAAATAATT
GTGGTGTTACGTCAAATGGTACAATTTATTGTATTGGGGGATATGATGGCTCTTCTATTATACCGAATGTAGAAGCA
TATGATCATCGTATGAAAGCATGGGTAGAGGTGGCACCTTTGAATACCCCTAGATCATCAGCTATGTGTGTTGCTTT
TGATAATAAAATTTATGTCATTGGTGGAACTAATGGTGAGAGATTAAATTCTATTGAAGTATATGAAGAAAAAATGA
ATAAATGGGAACAATTTCCATATGCCTTATTAGAAGCTAGAAGTTCAGGAGCAGCTTTTAATTACCTTAATCAAATA
TATGTTGTTGGAGGTATTGATAATGAACATAACATATTAGATTCCGTTGAACAATATCAACCATTTAATAAAAGATG
GCAATTTCTAAATGGTGTACCAGAGAAAAAAATGAATTTTGGAGCTGCCACATTGTCAGATTCTTATATAAT
I543T, total 603bp
TGGAAGTGCTGTATTGAATAATTTCTTATACGTTTTTGGTGGTAATAACTATGATTATAAGGCTTTATT
TGAAACTGAGGTGTATGATCGTTTAAGAGATGTATGGTATGTTTCAAGTAATTTAAATATACCTAGAAGAAATAATT
GTGGTGTTACGTCAAATGGTAGAATTTATTGTACTGGGGGATATGATGGCTCTTCTATTATACCGAATGTAGAAGCA
TATGATCATCGTATGAAAGCATGGGTAGAGGTGGCACCTTTGAATACCCCTAGATCATCAGCTATGTGTGTTGCTTT
TGATAATAAAATTTATGTCATTGGTGGAACTAATGGTGAGAGATTAAATTCTATTGAAGTATATGAAGAAAAAATGA
ATAAATGGGAACAATTTCCATATGCCTTATTAGAAGCTAGAAGTTCAGGAGCAGCTTTTAATTACCTTAATCAAATA
TATGTTGTTGGAGGTATTGATAATGAACATAACATATTAGATTCCGTTGAACAATATCAACCATTTAATAAAAGATG
GCAATTTCTAAATGGTGTACCAGAGAAAAAAATGAATTTTGGAGCTGCCACATTGTCAGATTCTTATATAAT
C580Y, total 603bp
TGGAAGTGCTGTATTGAATAATTTCTTATACGTTTTTGGTGGTAATAACTATGATTATAAGGCTTTATT
TGAAACTGAGGTGTATGATCGTTTAAGAGATGTATGGTATGTTTCAAGTAATTTAAATATACCTAGAAGAAATAATT
GTGGTGTTACGTCAAATGGTAGAATTTATTGTATTGGGGGATATGATGGCTCTTCTATTATACCGAATGTAGAAGCA
TATGATCATCGTATGAAAGCATGGGTAGAGGTGGCACCTTTGAATACCCCTAGATCATCAGCTATGTATGTTGCTTT
TGATAATAAAATTTATGTCATTGGTGGAACTAATGGTGAGAGATTAAATTCTATTGAAGTATATGAAGAAAAAATGA
ATAAATGGGAACAATTTCCATATGCCTTATTAGAAGCTAGAAGTTCAGGAGCAGCTTTTAATTACCTTAATCAAATA
TATGTTGTTGGAGGTATTGATAATGAACATAACATATTAGATTCCGTTGAACAATATCAACCATTTAATAAAAGATG
GCAATTTCTAAATGGTGTACCAGAGAAAAAAATGAATTTTGGAGCTGCCACATTGTCAGATTCTTATATAAT
The positive plasmid quality-control product and negative serum of having demarcated concentration in step 1) are configured according to certain concentration, added
The Proclion 300 for entering 0.05%, is dispensed according to 0.5ml specification, and -20 DEG C save backup.
9) specificity verification of four pairs of specific primers
It is mould using the positive sample containing synthetic plasmid in step 1) after four pairs of specific primer isoconcentrations are mixed
Plate, aqua sterilisa are that negative control carries out list primer single mode plate PCR reaction, verify the feasibility of specific primer, determine this experiment
Primer concentration and RT-PCR reaction condition used.
Shown in reaction system such as table 2, reaction condition is as shown in table 3:
2 PCR reaction system table of table
| PCR reaction reagent | Amount/hole (μ L) |
| 10X PCR Buffer | 2 |
| 25mM MgCl2 | 4 |
| Four species-specific primer F mixing | 2 |
| Four species-specific primer R mixing | 2 |
| Taq DNA Polymerase | 0.7 |
| DNA profiling | 4.3(20ng) |
| ddH2O | 5 |
| Total | 20 |
3 PCR reaction condition table of table
| Step | Temperature | Time |
| 1 | 94℃ | 10 minutes |
| 2 | 94℃ | 30 seconds |
| 3 | 54-62 DEG C of Gradient annealing | 30 seconds |
| 4 | 70℃ | 1 minute |
| 5 | N/A | Repeat 2-4 step 30 time |
| 6 | 4℃ | Continue: until collecting PCR product |
Take 10 μ LPCR to do agarose electrophoresis, Maker band be respectively from top to bottom 600bp, 500bp, 400bp, 300bp,
200bp and 100bp.Band 1-5 is respectively 54 DEG C, 56 DEG C, 58 DEG C, 60 DEG C, 62 DEG C of Gradient annealing temperature.Selection is suitably moved back
Fiery temperature.Experimental result is as follows: PCR reaction product electrophoretogram is as shown in Figs. 1-5；Wherein, Fig. 1 is R539T specific primer
PCR reaction product electrophoretogram；Fig. 2 is the PCR reaction product electrophoretogram of I543T specific primer；Fig. 3 is that C580Y specificity is drawn
The PCR reaction product electrophoretogram of object；Fig. 4 is the PCR reaction product electrophoretogram of Y493H specific primer, and Fig. 5 is four pairs of specificity
The PCR reaction product electrophoretogram of primer；
Each amplified fragments size of four pairs of specific primers is respectively as follows: C580Y specific primer 374bp, R539T in Fig. 5
Specific primer 113bp, I543T specific primer 232bp, Y493H specific primer 200bp；
Obtained by Fig. 1-Fig. 5 display: the specificity verification of four pairs of specific primers is as a result, it has been found that four pairs of specificity are drawn
The specificity of object is good, and band is single, without obvious miscellaneous band.Return of goods temperature is good in 54 DEG C -62 DEG C amplifications, determines that PCR expands
Increasing optimum annealing temperature is 56 DEG C.
10) feasibility of four pairs of primer amplifieds
Four pairs of specific primers are mixed with comparable sodium with universal primer, PCT primer mix is that all specificity are drawn
The mixing of upstream primer and downstream primer in object, the upstream primer of each pair of specific primer and the concentration of downstream primer are
200nM；Universal primermix is the mixing of the upstream primer and downstream primer in universal primer, universal primer it is upper
The concentration for swimming primer and downstream primer is 2 μM；
The positive sample of synthetic plasmid is template, and aqua sterilisa is that negative control carries out more primer multi-template PCR reactions, verifying
The feasibility of multiplex amplification system.
Reaction system is as shown in table 4, and reaction condition is as shown in table 5:
4 PCR reaction system table of table
| PCR reaction reagent | Amount/hole (μ L) |
| 10X PCR Buffer | 2 |
| 25mM MgCl2 | 4 |
| PCR primer mix | 2 |
| universal primer mix | 2 |
| Taq DNA Polymerase | 0.7 |
| DNA profiling | 9.3(50ng) |
| Total | 20 |
PCR reaction is carried out by temperature shown in table 5 after each substance in table 4 is mixed；
5 PCR reaction condition table of table
| Step | Temperature | Time |
| 1 | 94℃ | 10 minutes |
| 2 | 94℃ | 30 seconds |
| 3 | 60℃ | 30 seconds |
| 4 | 70℃ | 1 minute |
| 5 | N/A | Repeat 2-4 step 10 time |
| 6 | 94℃ | 30 seconds |
| 7 | 50℃ | 30 seconds |
| 8 | 70℃ | 1 minute |
| 9 | N/A | Repeat 6-8 step 10 time |
| 10 | 94℃ | 30 seconds |
| 11 | 50℃ | 30 seconds |
| 12 | 70℃ | 1 minute |
| 13 | N/A | Repeat 2-4 step 20 time |
| 14 | 4℃ | Continue: until collecting PCR product |
After PCR, PCR product and reagent are added in 96 hole sample panels in ratio shown in table 6.
6 reaction system of table
| GeXP sample | Amount/hole (μ L) |
| SLS sample solution | 38.5 |
| DNASize Standard-400 | 0.5 |
| PCR product | 1 |
| Total | 40 |
| Mineral oil | 1 drop |
Prepare separating liquid: about 220 μ L separating liquids are added on 96 hole separating liquid plates in an appropriate number of hole.Select " Frag-
3 " sample separation methods, (being detailed in GenomeLab GeXP genetic analyzer specification) interpretation of result (are detailed in GenomeLab
GeXP genetic analyzer specification).
A) plasmid for being separately added into a kind of mutated genes carries out more primer single mode plate GeXP as template, as a result such as Fig. 6
Shown in~9；
Wherein: Fig. 6 is the GeXP substance PCR testing result of R539T specific primer；Fig. 7 is Y493H specific primer
GeXP substance PCR testing result；Fig. 8 is the GeXP substance PCR testing result of I543T specific primer；Fig. 9 is that C580Y is special
The GeXP substance PCR testing result of property primer；
B) carry out more primer multi-templates of the plasmid of two kinds, three kinds, four kinds mutated genes as template are being added at random
GeXP, as a result as shown in Figure 10~15；
Wherein: Figure 10 is the GeXP multiplex PCR detection of the mixture of C580Y specific primer and R539T specific primer
As a result；Figure 11 is the GeXP multiplex PCR testing result of the mixture of R539T specific primer and Y493H specific primer；Figure 12
For the GeXP multiplex PCR testing result of R539T specific primer and the mixture of I543T specific primer；Figure 13 is C580Y special
The GeXP multiplex PCR testing result of the mixture of specific primer, R539T specific primer and Y493H specific primer；Figure 14
For the GeXP multiplex PCR detection of the mixture of I543T specific primer, R539T specific primer and Y493H specific primer
As a result；Figure 15 is I543T specific primer, R539T specific primer, Y493H specific primer and C580Y specific primer
Mixture GeXP multiplex PCR testing result；
From Fig. 6~15: the PCR product of the mixing plasmid containing four pairs of specific primers is 4 purpose bands, by 15
Figure is it is found that four peak values of specific amplification meet R539T positioned at being 113.24bp, 200.09bp, 232.14bp, 374.85bp
The specific amplified clip size of specific primer, Y493H specific primer, I543T specific primer and C580Y.
11) GeXP multi-PCR detection method stability experiment
A) source of people DNA is interfered
GD1 group, in the PCR system of 20 μ L, it is 5 × 10 that concentration, which is added,3Copy's is mixed containing four species-specific primers
Conjugative plasmid and 5ng people's complete genome DNA；GD2 group, in the PCR system of 20 μ L, it is 5 × 10 that concentration, which is added,3Copy's contains four
The mixing plasmid and 50ng people's complete genome DNA of species-specific primer；GD3 group, in the PCR system of 20 μ L, it is 5 that concentration, which is added,
×103The mixing plasmid and 500ng people's complete genome DNA containing four species-specific primers of copy.Every group is done 18 repetitions.
B) source of people RNA is interfered
GR1 group, in the PCR system of 20 μ L, it is 5 × 10 that concentration, which is added,3Copy's is mixed containing four species-specific primers
Conjugative plasmid and 500ng people RNA；
GR2 group, in the PCR system of 20 μ L, it is 5 × 10 that concentration, which is added,3Copy's is mixed containing four species-specific primers
Conjugative plasmid and 50ng people RNA；
GR3 group, in the PCR system of 20 μ L, it is 5 × 10 that concentration, which is added,3Copy's is mixed containing four species-specific primers
Conjugative plasmid and 5ng people RNA.Every group is done 18 repetitions, shown in measurement result table 7:
7 Detection of Stability result of table
By table 7 the results show that GD1 and GR1 do not influence the testing result to SNP, there are one by GD2, GD3, GR2 and GR3
Determine interference；Therefore, it needs to be added RNA enzyme appropriate when extracting DNA, removes RNA, while needing to control template in PCR system
Amount of DNA, total amount be no more than 50ng, avoid PCR amplification from being interfered.
C) detection limit determines
Detection limit range
Standard items are diluted to 5 × 10 with standard dilutions6Copy/ μ L, then does 10 doubling dilutions, and each gradient is dense
The sample of degree repeats 20 parts, and testing result is as shown in table 8, by the minimum standard product concentration with 90%-95% positive rate
As minimum detection limit.
Each Concentration Testing of table 8 limits result
As shown in Table 8, the detection of GeXP multiple PCR method is limited to 5 × 103copy/μL。
12) specificity of GeXP multiple PCR method
4 kinds of SNP mutations of K13 gene in plasmodium falciparum are detected, four groups of samples is separately designed, is tertian fever group, three respectively
Day malaria group, tertian fever+malignant malaria group and malarlae malaria+malignant malaria group, the sample of each concentration should do 3 multiple holes in every group, point
Not are as follows:
Group 1: Plasmodium vivax (Plasmodium vivax) DNA (5 × 106Copy), multiple for detecting GeXP
Whether PCR method is able to detect Plasmodium vivax DNA；
Group 2: malariae (Plamodiummalariae) DNA (5 × 106Copy), multiple for detecting GeXP
Whether PCR method is able to detect malariae DNA；
Group 3: wild type plasmodium falciparum DNA (5 × 106Copy), whether can for detecting GeXP multiple PCR method
Enough detect wild type plasmodium falciparum DNA；
Group 4: Plasmodium vivax DNA (5 × 103) and saltant type plasmodium falciparum copy
(Plamodiummalariae) synthetic plasmid DNA (5 × 103copy)；
Group 5: malariae DNA (5 × 103) and saltant type plasmodium falciparum copy
(Plamodiummalariae) synthetic plasmid DNA (5 × 103copy)；
Group 6: wild type plasmodium falciparum DNA (5 × 103) and saltant type plasmodium falciparum copy
(Plamodiummalariae) synthetic plasmid DNA (5 × 103copy)；
Testing result is as shown in table 9:
9 detection method specificity of table confirms result
As shown in Table 9, the specificity of GeXP multiple PCR method is good, can exclude Plasmodium vivax, malariae
DNA and wild type plasmodium falciparum.
13) detection performance
The detection performance verifying of GeXP multiple PCR method includes: positive coincidence rate, negative match-rate, total coincidence rate, sensitive
Degree, specificity and method efficiency.
Using Plasmid DNA standard items (4 parts), the clinical samples (8 parts), compound sample respectively containing four species-specific primers
(12 parts) and other plasmodium samples (4 parts), totally 28.Wherein positive clinical sample is by Chinese Center for Disease Control and Prevention parasitism
Worm is provided, including Y493H sudden change sample 2, and R539T sudden change sample 1, I543T sudden change sample 2, C580Y sudden change sample
3.All sample standard deviations are sent outside carry out gene sequencing, and the results are shown in Table 10,
10 clinical samples GeXP result of table
11 GeXP of table is compared with gene sequencing result
The diagnostic of 12 GeXP of table is evaluated
By table 10, it is found that the sensitivity of GeXP multiple PCR method is 100%, specificity is for table 11 and table 12
99.81%, accuracy 99.15%, compared with PCR sequencing PCR, testing result consistency is 99.32%.
14) clinical application
The plasmodium falciparum specimen dna detected in laboratory 2016-2018 entry and exit crowd is collected, totally 28, is applied
GeXP multiple PCR method detects plasmodium falciparum k13 resistant gene mutational site Y493H, R539T, I543T and C580Y, knot
Fruit is feminine gender.
Embodiment 2
A method of detection four resistance mutation sites of plasmodium falciparum k13 gene, this method are based on GeXP system
Realize the detection of four resistance mutation sites of plasmodium falciparum k13 gene；Four resistance mutation sites of plasmodium falciparum k13 gene
It is respectively as follows: Y493H, R539T, I543T and C580Y；
GeXP system combines multiple PCR technique and capillary electrophoresis technique；
This method specifically includes the following steps:
1) design primer group
The primer sets include a pair of of universal primer and for four pairs of plasmodium falciparum k13 four resistance mutation sites of gene
Specific primer, four pairs of specific primers are respectively as follows: Y493H specific primer, R539T specific primer, I543T specificity and draw
Object and C580Y specific primer；
Y493H specific primer is the end connection universal primer sequence composition in Y493H；R539T specific primer is
It is formed in the end connection universal primer sequence of R539T；I543T specific primer is the end connection universal primer in I543T
Sequence composition；C580Y specific primer is the end connection universal primer sequence composition in C580Y；
2) DNA of plasmodium falciparum is extracted
The DNA that plasmodium falciparum is extracted from the blood of human body needs that RNA first is added in the blood of human body before extracting
Enzyme；And the concentration of plasmodium falciparum is not less than 5 × 10 in the blood of human body of detection3copy/μL；
3) amplification system is established
Initiation polyad is combined by four pairs of specific primers described in step 1) and using the universal primer of fluorescent marker
System's amplification, specifically are as follows: by four pairs of specific primers described in step 1), using the universal primer and step 2) of fluorescent marker
In extracted malignant malaria original DNA mix to establish amplification system；
4) amplified production separates
Obtained product after step 3) amplification is subjected to capillary electrophoresis technique separation,
5) type of mutated gene is determined
The PCR product containing fluorescent marker after the completion of amplification in step 4) is detected and calculated through GeXP detection window
The size of amplified fragments compares the size for the amplified fragments that the size of amplified fragments obtains after quality-control product amplification to really
Determine the type of mutated gene.
The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Claims (5)
1. a kind of method for detecting four resistance mutation sites of plasmodium falciparum k13 gene, which is characterized in that this method is to be based on
The detection of GeXP system realization four resistance mutation sites of plasmodium falciparum k13 gene；Four resistances of plasmodium falciparum k13 gene
Mutational site is respectively as follows: Y493H, R539T, I543T and C580Y；
GeXP system combines multiple PCR technique and capillary electrophoresis technique；
This method specifically includes the following steps:
1) design primer group
The primer sets include a pair of of universal primer and special for four pairs of plasmodium falciparum k13 four resistance mutation sites of gene
Property primer, four pairs of specific primers be respectively as follows: Y493H specific primer, R539T specific primer, I543T specific primer with
And C580Y specific primer；
Y493H specific primer is the end connection universal primer sequence composition in Y493H；R539T specific primer be
The end connection universal primer sequence of R539T forms；I543T specific primer is the end connection universal primer sequence in I543T
Column composition；C580Y specific primer is the end connection universal primer sequence composition in C580Y；
2) DNA of plasmodium falciparum is extracted
The DNA that plasmodium falciparum is extracted from the blood of human body needs that RNA enzyme first is added in the blood of human body before extracting；And
The concentration of plasmodium falciparum is not less than 5 × 10 in the blood of human body of detection3copy/μL；
3) amplification system is established
It is combined four pairs of specific primers described in step 1) and using the universal primer of fluorescent marker and causes multiple system expansion
Increase, specifically are as follows: by four pairs of specific primers described in step 1), using institute in the universal primer and step 2) of fluorescent marker
The DNA of the malignant malaria original of extraction mixes to establish amplification system；
4) amplified production separates
Obtained product after step 3) amplification is subjected to capillary electrophoresis technique separation,
5) type of mutated gene is determined
The PCR product containing fluorescent marker after the completion of amplification in step 4) is detected through GeXP detection window and calculates amplification
The size of segment compares the size for the amplified fragments that the size of amplified fragments obtains after quality-control product amplification so that it is determined that prominent
Become the type of gene.
2. a kind of method for detecting four resistance mutation sites of plasmodium falciparum k13 gene according to claim 1, special
Sign is, the forward primer sequence of the Y493H specific primer are as follows: AGGTGACACTATAGAATATGGAAGTGCTGTATT
GAATAATTTCTTACAC,
The reverse primer sequences of the Y493H specific primer are as follows: GTACGACTCACTATAGGGAATTTGACGTAACACCAC
AATTATTTCTTCTAG；
The forward primer sequence of the R539T specific primer are as follows: AGGTGACACTATAGAATATAATTGTGGTGTTACGTC
AAATGGTACA；
The reverse primer sequences of the R539T specific primer are as follows: GTACGACTCACTATAGGGATACATTCGGTATAATAG
AAGAGCCATCATA；
The forward primer sequence of the I543T specific primer are as follows: AGGTGACACTATAGAATATGGTGTTACGTCAAATGG
TAGAATTTATTGTACT；
The reverse primer sequences of the I543T specific primer are as follows: GTACGACTCACTATAGGGACTCTCACCATTAGTTCC
ACCAATGAC；
The forward primer sequence of the C580Y specific primer are as follows: AGGTGACACTATAGAATAATACCCCTAGATCATCAG
CTATGTGT；
The reverse primer sequences of the C580Y specific primer are as follows: GTACGACTCACTATAGGGAATTATATAAGAATCTGA
CAATGTGGCAGCT。
3. a kind of method for detecting four resistance mutation sites of plasmodium falciparum k13 gene according to claim 1, special
Sign is that the universal primer includes upstream primer and downstream primer, the primer sequence of upstream primer are as follows:
AGGTGACACTATAGAATA, the primer sequence of downstream primer are as follows: GTACGACTCACTATAGGGA.
4. a kind of detection four resistance mutation sites of plasmodium falciparum k13 gene according to claim 1-3
Method, which is characterized in that the specific amplified clip size of the Y493H is 200bp；The specific amplified segment of the R539T is big
Small is 113bp；The specific amplified clip size of the I543T is 232bp；The specific amplified clip size of the C580Y is
374bp。
5. a kind of method for detecting four resistance mutation sites of plasmodium falciparum k13 gene according to claim 4, special
Sign is, is the length that amplified fragments are calculated according to detection segment and standard molecule segment transit time in step 4).
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| Title |
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| 胡凌: ""GeXP 多重基因表达分析系统研究进展"", 《ANIMAL HUSBANDRY ＆ VETERINARY MEDICINE》 * |
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Application publication date: 20190524 |