CN107299154A - Dual PCR (polymerase chain reaction) special primer for sheep theileria luwenshuni and phagocytophilic cell anaplasma and dual PCR detection method - Google Patents

Dual PCR (polymerase chain reaction) special primer for sheep theileria luwenshuni and phagocytophilic cell anaplasma and dual PCR detection method Download PDF

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CN107299154A
CN107299154A CN201710559840.5A CN201710559840A CN107299154A CN 107299154 A CN107299154 A CN 107299154A CN 201710559840 A CN201710559840 A CN 201710559840A CN 107299154 A CN107299154 A CN 107299154A
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theileria
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宁长申
闫亚群
崔艳艳
赵姗姗
王晓星
张艳
史柯
菅复春
张龙现
王荣军
景纪春
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Henan Agricultural University
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Abstract

The invention discloses a sheep Theileria luwenshuni and phagocytophile anaplasma PCR special primer, the nucleotide sequence of the primer is: special primer T.lu7 for Theileria lanuginosa: as shown in SEQ NO.1 and SEQ NO. 2; the primer SSAP2 special for the anaplasma phagocytophile: shown as SEQ NO.3 and SEQ NO. 4. The size of the amplified product of the T.lu7 special primer for theileria foetida is 962bp, and the size of the amplified product of the SSAP2 special primer for anaplasma phagocytophilum is 641 bp. The invention is based onT. luwenshuni18S rRNA target gene andA. phagocytophilumthe double PCR method established by the 16S rRNA target gene has high specificity. The method has the advantages of high detection speed, high sensitivity, strong specificity, high efficiency, low cost and the like, and is suitable for epidemiological investigation and clinical confirmation of diseases caused by two pathogens.

Description

A kind of sheep Theileria luwenshuni and Anaplasma phagocytophilum double PCR primer special and Dual PCR detection method
Technical field
The invention belongs to technical field of biological, and in particular to a kind of sheep Theileria luwenshuni and Anaplasma phagocytophilum Double PCR primer special and dual PCR detection method.
Background technology
Theileriosis in sheep (Ovine And CaprineTheileriosis) is that the protozoon belonged to by Taylor section Taylor parasitizes The general name of caused disease in sheep and goat macrophage, lymphocyte and red blood cell, belongs to one kind and is propagated by Tick victor Tick transmissibility bloodprotozoonoses (Neitz W O 1957, Barnett S F 1977, Morel P C et al, 1981).Taylor Worm is found (Littlewood 1915) in first time in 1914 in Egypt, and China was most reported earlier than 1958 by Yang Fuguo etc. The road theileriosis in sheep in Sichuan (Yang et al, 1958).The disease can cause goat and sheep heating, anemia and jaundice etc. Clinical symptoms, make sheep hypoevolutism, and meat yield and wool production are remarkably decreased, and lamb and external sheep can be caused only a large amount of dead Die, so as to limit the development and production of region sheep husbandry, up to the present, existing six kinds of Theileria SPs are reported, respectively For the stronger Taylor Lai Shi worm (Theilerialestoquardi) of pathogenicity, Theileria luwenshuni (T.luwenshuni) and You Shi Taylor worm (T.uilenbergi), and to sheep pathogenicity is very weak or continuous Theileria SP (T.ovis) of no pathogenicity, separation are safe Strangle worm (T.separata) and hiding Taylor worm (T.recondita).Wherein T.luwenshuni, T.uilenbergi and T.ovis is found at home.Root is it is reported that the goat and sheep of China southeast, the Northwest and middle part have T.luwenshuni infects.
Anaplasma phagocytophilum (A.phagocytophilum, AP) was found first in 1932 in Scotland area, The not clear heating of sheep is caused, because it is found that the disease is relevant with the tick worm in herbage, ticks heat transfer is referred to as sick (TBF).By Human granulocytic anaplasmosis caused by A.phagocytophilum (human granulocytic anaplasmosis, HGA) It is Amphixenosis, the initial cause of disease is referred to as Human Granulocytic Ehrlichia, is reclassified belong to Richettsia later Mesh, no slurry section, no slurry category is renamed as human granular leukocyte without slurry (A.phagocytophilum), A.phagocytophilum has extensive host group, including the mankind, wild animal and domestic animal etc., is had to humans and animals Relatively strong pathogenic infecting both domestic animals and human pathogen, belongs to disease of natural focus, can cause the acute and chronic infection of ruminant Disease, A.phagocytophilum can induce body to produce inflammatory reaction and immune response, mediated immunity competent cell and inflammation immediately Attack of the sex factor to host cell, ultimately results in various scabies secondary infections and organ caused by immunosupress and potential disease and declines Exhaust, ill feature is mainly shown as gollbladder dilation, anaemia, jaundice, hyperpyrexia, become thin, and severe patient even can cause death.
A.phagocytophilum is as a kind of people and Amphixenosis's cause of disease of many animals, with public hygienics Meaning, is paid much attention to by people, and the generation of Anaplasma phagocytophilum disease is in season distribution, occurs mainly in the hard tick of medium During activity, in selected areas of China, hard tick, dermacentor silvarum, haemaphysalis longicornis etc. are all the propagation matchmakers of Anaplasma phagocytophila It is situated between, susceptible animal and people are bitten through the young tick of infection and cause transmission of infection.China finds HGA disease diseases in 2006 in Anhui Province's report Example, hereafter saved in Shandong, Hubei, Henan, Zhejiang etc. has morbidity to report successively, is all detected in saving domestic animal in Anhui, Hubei etc. A.phagocytophilum, also there is the animal infection sick relevant report in Yili of Xinjiang.
The content of the invention
The purpose of the present invention is in view of the shortcomings of the prior art there is provided a kind of sheep Theileria luwenshuni and thermophilic phagocyte without slurry Body double PCR primer special and dual PCR detection method.
The purpose of the present invention is realized in the following manner：
A kind of sheep Theileria luwenshuni and Anaplasma phagocytophilum double PCR primer special, the nucleotides sequence of primer is classified as：
Sheep Theileria luwenshuni primer special T.lu7：
T.lu7F：5 '-TGCTGCATTGCATCTTCT-3 ', as shown in SEQ NO.1；
T.lu7R：5 '-AGGGACGTAATCTGCACAAG-3 ', as shown in SEQ NO.2；
Anaplasma phagocytophilum primer special SSAP2：
SSAP2F：5 '-GCTGAATGTGGGGATAATTTAT-3 ', as shown in SEQ NO.3；
SSAP2R：5 '-ATGGCTGCTTCCTTTCGGTTA-3 ', as shown in SEQ NO.4.
The amplified production size of the sheep Theileria luwenshuni primer special T.lu7 is 962bp, and Anaplasma phagocytophilum is special It is 641bp with primer SSAP2 amplified production size.
Double PCR inspection is carried out using above-mentioned sheep Theileria luwenshuni and Anaplasma phagocytophilum double PCR primer special The method of survey, comprises the following steps：
(1) testing sample DNA extraction；
(2) primer pair T.lu7 and SSAP2 are each configured to the solution that concentration is 25 μM/L；
(3) double PCR amplification is carried out by template of testing sample DNA：
PCR25 μ L amplification reaction systems are：The μ L of 0.25 μ L, 10 × LA Buffer of LA Taq DNA Polymeras 3.0, Each each 0.5 μ L of 0.5 μ L, SSAP2 F and SSAP2 R of dNTP Mixture 4.0 μ L, T.lu7 F and T.lu7 R, DNA profiling 2.0 μ L, sterile deionized water complements to 25 μ L；
Pcr amplification reaction condition is：94 DEG C of pre-degenerations 5min, 94 DEG C of denaturation 35s, 55 DEG C of annealing 35s, 72 DEG C of extension 35s, operation 40 circulations, 72 DEG C of extension 10min；
(4) decision method of amplified production：After PCR reaction terminatings, 5 μ L PCR primers are taken, is detected and expanded with 1% agarose electrophoresis As a result：Amplification obtains 962bp sizes product and is then judged in sample containing sheep Theileria luwenshuni, and amplification obtains the big abortions of 641bp Thing is then judged in sample containing Anaplasma phagocytophilum.
Relative to prior art, the present invention
(1) it is simple to operate；Two kinds of cause of diseases can be detected simultaneously by only needing a reactant system to carry out once amplification, time saving and energy saving.
(2) result judgement is simple；Occur two purpose bands of 962bp, 641bp in agarose gel electrophoresis, can determine whether sense Contaminate two kinds of cause of diseases；Only there is a purpose band to can determine whether to have infected a certain cause of disease：T.luwenshuni (962bp) or A.phagocytophilum(641bp)。
(3) present invention is based on T.luwenshuni18S rRNA target genes and A.phagocytophilum16S rRNA targets The dual-PCR method specificity that gene is set up is high.The method has detection speed fast, and sensitiveness is high, high specificity, it is efficient, low into This advantages of, epidemiology survey and clinical definite suitable for two kinds of cause of disease diseases caused.
Brief description of the drawings
Fig. 1 is different LA Taq enzymes amount double PCR amplifications：
M:DL2000Maker；1-9：LA Taq enzyme amounts are followed successively by:0.01μL、0.1μL、0.15μL、0.2μL、0.25μL、0.3μ L、0.35μL、0.4μL、0.45μL；10：Negative control.
Fig. 2 is difference 10 × LA Buffer amount double PCR amplifications：
M:DL2000Maker；1-9：10 × LA Buffer amounts are followed successively by 0.5 μ L, 1 μ L, 1.5 μ L, 2 μ L, 2.5 μ L, 3 μ L, 3.5 μ L、4μL、4.5μL；10：Negative control.
Fig. 3 is different dNTP Mixture amounts double PCR amplifications：
M:DL2000Maker；1-9：DNTP Mixture amounts are followed successively by 0.5 μ L, 1.5 μ L, 2 μ L, 2.5 μ L, 3 μ L, 3.5 μ L, 4 μ L、4.5μL、5μL；10：Negative control.
Fig. 4 is different primers amount double PCR amplification：
M:DL2000Maker；1-6：Primer proportioning is followed successively by 0.4:0.4、0.45:0.55、0.5:0.55、0.5:0.5、0.47: 0.53；7：Negative control.
Fig. 5 is different cycle-index double PCR amplifications：
M:DL2000Maker；1-5：Loop parameter is respectively 20,25,30,35,40；6：Negative control.
Fig. 6 is different temperatures gradient double PCR amplification：
M:DL2000Maker；1-12：Annealing temperature is respectively 51 DEG C, 52 DEG C, 53 DEG C, 54 DEG C, 55 DEG C, 56 DEG C, 57 DEG C, 58 DEG C, 59℃、60℃、61℃、62℃；13：Negative control.
Fig. 7 is double PCR specific test result：
M.DNA standards DL2000：1.T.luwenshuni+AP；2.T.luwenshuni；3.AP；4.T.ovis； 5.T.uilenbergi；6.T.annulata；7.A.ovis；8.A.bovis；9.A.marginale；10.B.motasi； 11.T.gondii；12.DdH2O.
Fig. 8 is T.luwenshuni sensitivity tests：
M:DL2000Maker；1-9 sample extension rates are：1、10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8；10. Negative control.
Fig. 9 is A.phagocytophilum sensitivity tests：
M:DL2000Maker；1-13 sample extension rates are：1、10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8、10-9、10-10、10-11、10-12；14. negative control.
Figure 10 is double PCR sensitivity tests：
M:DL2000Maker；1-13 sample extension rates are：1、10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8、10-9、10-10、10-11、10-12；14. negative control.
Figure 11 is replica test double PCR amplification：
M:DL2000Maker；1-3. it is repeated；4. negative control.
Figure 12 is dual PCR detection method PCR amplifications：
M:DL2000Maker；1-6.PCR product；7. positive control；8. negative control.
Figure 13 is primer T.lu7F/T.lu7RPCR amplification T.luwenshuni 18S rRNA genetic results：
M:DL2000Maker；1-6.PCR product.
Figure 14 is primer SSAP2f/SSAP2r PCR amplification A.phagocytophilum16S rRNA genetic results：
M:DL2000Maker；1-5.PCR product；6. positive control；7. negative control.
Embodiment
The present invention is further elaborated with reference to embodiments, facilitates a better understanding of the present invention, but be not intended to limit this Invention.
The design and dual PCR detection method of sheep Theileria luwenshuni and Anaplasma phagocytophilum double PCR primer special Research
1. design of primers and synthesis
Using the sequence analysis softwares of Clustal X 2.1, by the sheep Theilerialuwenshuni 18S rRNA in GenBank Sequence (accession number：KC735166.1) it is compared, is set with the softwares of Primer Premier 5.0 with other species homologous sequences 1 couple of species-specific primer T.lu7F/T.lu7R is counted, Sheng Gong bioengineering limited company synthesizes by Shanghai；Quote amplification A.phagocytophilum species-specific primers SSAP2f/SSAP2r (Kawahara Met al., 2006), primer details are shown in Table 1.
The double PCR primer sequence of table 1 and amplified production size
2.PCR reaction systems and reaction condition optimization
Optium concentration to the enzyme in PCR reaction systems, PCR Buffer, dNTP Mixture and primer is optimized；And it is right Optimum cycle parameter is optimized.Reaction system and condition are shown in Table 2-3.
The μ L amplification reaction systems of 2 double PCR of table 25
The double PCR amplification reaction condition of table 3
The optimization of LA Taq DNA Polymerase amounts：In the case of other conditions identical, in 25 μ L reaction systems In, LA Taq DNA Polymerase (5U/ μ L) dosage is respectively 0.01,0.1,0.15,0.2,0.25,0.3,0.35,0.4, 0.45 μ L enter performing PCR amplification.As a result optimum add amount is 0.25 μ L, the low influence synthetic product amount of concentration (see Fig. 1).
The optimization of 10 × LA Buffer amounts：In the case of other conditions identical, in 25 μ L reaction systems, 10 × LAPCR Buffer dosages are respectively that 0.5,1,1.5,2,2.5,3,3.5,4,4.5 μ L enter performing PCR amplification.As a result optimum content is 3 μ L, too low, influence PCR efficiency (see Fig. 2).
The optimization of dNTPMixture amounts：In the case of other conditions identical, in 25 μ L reaction systems, 2.5mM's DNTP Mixture dosages are respectively that 0.5,1.5,2,2.5,3,3.5,4,4.5,5 μ L enter performing PCR amplification.As a result dNTP When Mixture content is 4 μ L, expanding effect is preferably (see Fig. 3).
Optimization to primer in system：In the case of other conditions identical, in 25 μ L reaction systems, concentration is 25 μ M/L primer T.lu7F/T.lu7R and SSAP2f/SSAP2r dosage proportioning is respectively (μ L)：0.4:0.4；0.4:0.6；0.45: 0.55；0.5:0.55；0.5:0.5；0.47:0.53 enters performing PCR amplification.As a result primer T.lu7F/T.lu7 content is 0.5 μ L, SSAP2f/SSAP2r content is 0.5 μ L, and expanding effect is preferably (see Fig. 4).
Optimization to loop parameter：In the case of other conditions identical, in 25 μ L reaction systems, loop parameter difference Enter performing PCR amplification for 20,25,30,35,40.As a result 40 circulations are the most suitable loop parameter of the reaction, with the increasing of cycle-index Plus, the purpose band of amplification gradually blast (see Fig. 5).
The screening of most suitable annealing temperature：Positive to Theileria luwenshuni in Anaplasma phagocytophilum DNA carries out thermograde Double PCR expand, thermograde be 51 DEG C, 52 DEG C, 53 DEG C, 54 DEG C, 55 DEG C, 56 DEG C, 57 DEG C, 58 DEG C, 59 DEG C, 60 DEG C, 61 DEG C, 62℃.According to the brightness of purpose band under different annealing temperature and whether there is non-specific band and cross-dimerization body and carry out total score Analysis, determines the most suitable annealing temperature of the reaction system.As a result 51 DEG C -62 DEG C have specific band amplification, using 55 DEG C as anti- Answer the most suitable annealing temperature of PCR in parameter (see Fig. 6)
3. the recovery of product, clone and sequencing
PCR primer is reclaimed with DNA gel QIAquick Gel Extraction Kit, recovery product is connected with pMDl8-T carriers, Escherichia coli are transferred to In DH50 α competent cells, obtain positive bacterial plaque through the screening of blue hickie and expand culture, positive bacterium solution delivers to raw work bioengineering (Shanghai) limited company is sequenced.Using the softwares of DNAStar 7.1 and BLAST instruments by the nucleotide sequence measured with Correlated series in GenBank carries out tetraploid rice.As a result it will log in sheep Theileria luwenshuni sequencing result and GenBank The homology of related reference sequences is 99.4%-99.9%.Logged in Anaplasma phagocytophilum sequencing result and GenBank The homology of related reference sequences is 99.8%-100%.
4. specific test
Extract respectively Theileria luwenshuni, continuous Theileria SP, T.uilenbergi, annular loop detector, sheep without slurry, anaplasma bovis, Edge without slurry, Mohs Babesia, Infection of Toxoplasma Gondii genomic DNA, be used as the template of specific test.By optimal reaction system And condition enters performing PCR amplification, to examine the specificity of this method.As a result the double PCR system and reaction condition to have optimized, enter There is 962bp or so band, Anaplasma phagocytophilum genome in row specific test, sheep Theileria luwenshuni genomic DNA There is 641bp or so band, and continuous Theileria SP, T.uilenbergi, annular loop detector, sheep are without slurry, anaplasma bovis, side Edge does not occur band then without slurry, Mohs Babesia, Infection of Toxoplasma Gondii (see Fig. 7).
5. sensitivity tests
(1) sheep Theileria luwenshuni list PCR sensitivity tests
It is 291.3ng μ L to determine the sheep Theileria luwenshuni plasmid extracted as standard form concentration with ultraviolet specrophotometer-1, D260/D280=1.89 illustrates that extracted DNA pollutes without albumen and RNA, standard form pressed into 1：10 ratios are continuously dilute Release, enter performing PCR amplification by optimal reaction system and condition, detect most low DNA detection limit.As a result show, PCR can detect sheep Theileria luwenshuni DNA maximum dilution multiple is 1 × 10-7, i.e., minimum detectable 29.13fg μ L-1(see Fig. 8).
(2) Anaplasma phagocytophilum list PCR sensitivity tests
The Anaplasma phagocytophilum plasmid extracted is determined with ultraviolet specrophotometer and is used as standard form concentration 153.4ng μ L-1, D260/D280=1.89 illustrates that extracted DNA pollutes without albumen and RNA, standard form pressed into 1：10 ratios are continuously dilute Release, enter performing PCR amplification by optimal reaction system and condition, detect most low DNA detection limit.As a result show, PCR can detect thermophilic It is 1 × 10 to swallow the maximum dilution multiple without slurry DNA-10, i.e., minimum detectable 15.34ag μ L-1(see Fig. 9).
(3) dual PCR detection method sensitivity tests
Enter sheep Theileria luwenshuni and thermophilic phagocytosis in performing PCR amplification, detection double PCR by double PCR optimal reaction system and condition Cell is without slurry most low DNA detection limit.As a result show, the maximum dilution multiple that PCR can detect sheep Theileria luwenshuni DNA is 1×10-7, i.e., minimum detectable 29.13fg μ L-1；Thermophilic maximum dilution multiple of the phagocytosis without slurry DNA is 1 × 10-8, i.e., Minimum detectable 1.534fg μ L-1(see Figure 10).
6. replica test
Using sheep Theileria luwenshuni positive and Anaplasma phagocytophilum positive as template, reacted with the PCR optimized System and condition carry out 3 repetitions and detected, the stability of inspection institute's method for building up.As a result set up PCR method is used, with sheep Lv Taylor family name worm positive and Anaplasma phagocytophilum DNA profiling carry out 3 repetitions and detected, it is amplifiable go out specific fragment (see Figure 11).
7. clinical practice
The method set up with this experiment is entered performing PCR to 54 parts of sheep blood DNA samples for picking up from Luoyang Yiyang Zhao Baoxiang and detected, single PCR amplification T.luwenshuni positive rates are 85.19% (46/54), and A.phagocytophilum positive rates are 83.33% (45/54), mixed infection positive rate is 72.22% (39/54)；Double PCR amplification T.luwenshuni positive rates are 87.04% (47/54), A.phagocytophilum positive rates are 55.56% (30/54), and mixed infection positive rate is 50% (27/54)； Double PCR primer, reaction system and reaction condition are shown in Table 1,2,3 respectively, and detection statistics the results are shown in Table 4；Part picture see Figure 12- 14。
The double PCR of table 4 and single PCR detection method clinical detection result
0.01 ＜ P ＜ 0.05.
SEQUENCE LISTING
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Claims (3)

1. a kind of sheep Theileria luwenshuni and Anaplasma phagocytophilum double PCR primer special, it is characterised in that：The nucleosides of primer Acid sequence is：
Sheep Theileria luwenshuni primer special T.lu7：
T.lu7F：5 '-TGCTGCATTGCATCTTCT-3 ',
T.lu7R：5’-AGGGACGTAATCTGCACAAG-3’；
Anaplasma phagocytophilum primer special SSAP2：
SSAP2F：5 '-GCTGAATGTGGGGATAATTTAT-3 ',
SSAP2R：5’-ATGGCTGCTTCCTTTCGGTTA-3’.
2. sheep Theileria luwenshuni according to claim 1 and Anaplasma phagocytophilum double PCR primer special, its feature It is：The amplified production size of the sheep Theileria luwenshuni primer special T.lu7 is 962bp, and Anaplasma phagocytophilum is special Primer SSAP2 amplified production size is 641bp.
3. carried out using the sheep Theileria luwenshuni described in claim 1 and Anaplasma phagocytophilum double PCR primer special double The method of weight PCR detections, it is characterised in that：Comprise the following steps：
(1) testing sample DNA extraction；
(2) primer pair T.lu7 and SSAP2 are each configured to the solution that concentration is 25 μM/L；
(3) double PCR amplification is carried out by template of testing sample DNA：
PCR25 μ L amplification reaction systems are：The μ L of 0.25 μ L, 10 × LA Buffer of LA Taq DNA Polymeras 3.0, Each each 0.5 μ L of 0.5 μ L, SSAP2 F and SSAP2 R of dNTP Mixture 4.0 μ L, T.lu7 F and T.lu7 R, DNA profiling 2.0 μ L, sterile deionized water complements to 25 μ L；
Pcr amplification reaction condition is：94 DEG C of pre-degenerations 5min, 94 DEG C of denaturation 35s, 55 DEG C of annealing 35s, 72 DEG C of extension 35s, operation 40 circulations, 72 DEG C of extension 10min；
(4) decision method of amplified production：After PCR reaction terminatings, 5 μ L PCR primers are taken, is detected and expanded with 1% agarose electrophoresis As a result：Amplification obtains 962bp sizes product and is then judged in sample containing sheep Theileria luwenshuni, and amplification obtains the big abortions of 641bp Thing is then judged in sample containing Anaplasma phagocytophilum.
CN201710559840.5A 2017-07-11 2017-07-11 Dual PCR (polymerase chain reaction) special primer for sheep theileria luwenshuni and phagocytophilic cell anaplasma and dual PCR detection method Pending CN107299154A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109913566A (en) * 2019-04-18 2019-06-21 河南农业大学 The combination of Quadruple- PCR primer special and Quadruple- PCR detection method for four kinds of detection simultaneously without slurry
CN110283922A (en) * 2019-07-01 2019-09-27 海南大学 It is a kind of for detecting the PCR primer, detection reagent and detection method of sheep Theileria luwenshuni

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CN105154557A (en) * 2015-09-22 2015-12-16 河南农业大学 Dual PCR method for detecting theileria hirci and anaplasma
CN106435025A (en) * 2016-09-28 2017-02-22 福建省农业科学院畜牧兽医研究所 Primer special for dual PCR of two sheep causative agents and dual PCR detection method

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CN105154557A (en) * 2015-09-22 2015-12-16 河南农业大学 Dual PCR method for detecting theileria hirci and anaplasma
CN106435025A (en) * 2016-09-28 2017-02-22 福建省农业科学院畜牧兽医研究所 Primer special for dual PCR of two sheep causative agents and dual PCR detection method

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109913566A (en) * 2019-04-18 2019-06-21 河南农业大学 The combination of Quadruple- PCR primer special and Quadruple- PCR detection method for four kinds of detection simultaneously without slurry
CN110283922A (en) * 2019-07-01 2019-09-27 海南大学 It is a kind of for detecting the PCR primer, detection reagent and detection method of sheep Theileria luwenshuni

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Application publication date: 20171027