CN107227350A - A kind of probe groups, kit and the method for the fracture of quick detection ALK gene - Google Patents

A kind of probe groups, kit and the method for the fracture of quick detection ALK gene Download PDF

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CN107227350A
CN107227350A CN201710431520.1A CN201710431520A CN107227350A CN 107227350 A CN107227350 A CN 107227350A CN 201710431520 A CN201710431520 A CN 201710431520A CN 107227350 A CN107227350 A CN 107227350A
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probe
fluorescence
amplification
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alk gene
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方国伟
洪冉
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DMD BIOMED Ltd
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q2600/00—Oligonucleotides characterized by their use
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Abstract

The invention discloses a kind of method of use fluorescence in situ hybridization technique quick detection ALK gene fracture, it is characterised in that the detection of each target site needs three kinds of probes, is respectively：Contact probe, amplification probe and fluorescence probe.Wherein combined with ALK gene broken site two ends, there is the repetitive sequence of 20 group of 20 base at 3 ' ends, specifically can be combined with amplification probe 5 ' ends of contact probe；Hold 5 ' ends of amplification probe and combine with the 3 ' of contact probe, there is the repetitive sequence of 20 group of 20 base at 3 ' ends, specifically can be combined with fluorescence probe；5 ' ends of fluorescence probe are marked with CY3 or FITC, and by the synergy of these three probes, fluorescence signal is amplified into 400 times, realize that ALK gene is broken quick, sensitive detection.The invention also discloses a kind of kit of quick detection ALK gene fracture and probe groups.

Description

A kind of probe groups, kit and the method for the fracture of quick detection ALK gene
Technical field
The invention belongs to biology field, more particularly to a kind of probe groups and kit, by using fluorescent in situ ALK gene fracture in the means of hybridization, quick detection sample.
Background technology
Lung cancer is China or even Global mortality highest malignant tumour, wherein non-small cell lung cancer (Non-Small Cell Lung Cancer, NSCLC) it is most common hypotype, account for the 85% of lung cancer morbidity rate.In recent years, with to NSCLC The further investigation of molecular biological characteristics, when advanced NSCLC has progressed into the individualized treatment that targeting driving gene is instructed Generation.Positive NSCLC molecular isoforms are broken especially for ALK gene.Anaplastic lymphoma kinase (anaplasticlymphoma kinase, ALK) gene break positive is a NSCLC specific molecular hypotype, is accounted for 5% or so of NSCLC.ALK is a kind of receptor tyrosine kinase, is insulin receptor (IR) superfamily member.ALK is mainly expressed Do not expressed in developmental maincenter and peripheral neverous system, the tissue such as the normal lung in adult, illustrate ALK to nervous system Normal development and Function effect.Although ALK normal physiological function is not yet illustrated completely, work as itself and other bases Expression is reset because occurring fusion, then the carcinogenicity with height.Gram azoles can be broken the NSCLC triggered for medicines such as Buddhist nuns to ALK Carry out specific targeted therapy.
For these reasons, ALK gene fracture is put into the clinical essential items for inspection of NSCLC, and ALK gene is clinically detected at present The common methods of fracture have SABC (IHC) and FISH (FISH), and wherein FISH is detection ALK gene fracture Goldstandard.But tradition FISH uses genomic probe, probe total length reaches more than 100Kb, because probe length is extremely long, covered Lid scope is very big so that non-specific hybridization inevitably occurs in partial sequence, causes background higher；Meanwhile, long spy Pin also often results in the diverging of fluorescence signal point, is unfavorable for confirmation and the statistics of signal.Due to traditional FISH probe unit length Fluorescence intensity is limited, is solved the above problems with the method for probe length is reduced, fluorescence signal intensity can be reduced, be unfavorable for Detection.
The content of the invention
It is an object of the invention to provide a kind of probe groups, by the means of FISH, a kind of quick, spirit is set up Method that is quick, specifically detecting ALK gene fracture in sample.
The method of the quick detection ALK gene fracture of the present invention is compared with traditional FISH methods, signaling point low with background Concentrate, high specificity, the advantages of speed is fast, can fast and efficiently detect that ALK gene is broken.
The present invention is achieved by the following technical solutions：
The invention provides a kind of probe groups of quick detection ALK gene fracture, the probe groups are by contact probe, amplification Three kinds of probe compositions of probe and fluorescence probe, the contact probe includes contact probe 1 and contact probe 2, the amplification probe Including amplification probe 1 and amplification probe 2, the fluorescence probe includes fluorescence probe 1 and fluorescence probe 2, wherein, contact probe 1, Amplification probe 1, fluorescence probe 1, contact probe 2, the base sequence of amplification probe 2 and fluorescence probe 2 are respectively：
Contact probe 1：
5’-TAGTTGGTAAGGTAACGGCTTACCAAGGCA(GTATJCGCJCTGFTATJCCG)-3’；
Amplification probe 1：
5’-CGGFATAJCAGFGCGFATAC(TCFACGJCFCTAJGGAFAAFG)-3’；
Fluorescence probe 1：5’-CY3-JTTJTCCFTAGJGFCGTJGA-3’；
Contact probe 2：
5’-AGGCGACTTTCTGGTCTGTAACTGACGCTG(AGTFAJCGCFGTAFCAAJTJ)-3’；
Amplification probe 2：
5’-FAFTTGJTACJGCGFTJACT(GCAFJTTACCGFAJJTACFT)-3’；
Fluorescence probe 2：5’-FITC-AJGTAFFTJCGGTAAFJTGC-3’.
Preferably, 5 ' ends of the fluorescence probe 1 and the fluorescence probe 2 are marked with fluorophor.
Preferably, the fluorophor is CY3, FITC or FAM.
Further, the contact probe 1, the amplification probe 1 and the fluorescence probe 1 are used to detect that ALK gene is broken Split site N-terminal, ALK gene broken site N-terminal is labeled as red fluorescence by the fluorescence probe 1, it is the contact probe 2, described Amplification probe 2 and the fluorescence probe 2 are used to detect ALK gene broken site C-terminal, and ALK gene is broken by the fluorescence probe 2 Site N-terminal is labeled as the F in green fluorescence, the base sequence of the contact probe, the amplification probe and the fluorescence probe Represent that the C methylated, J represent the G methylated, be introduced into non-existent base in two kinds of natural gene group DNA of F/J, can be effective Ground reduces non-specific hybridization, improves the specificity of detection.
Present invention also offers a kind of kit being broken for quick detection ALK gene, the kit includes hybridization Liquid and the above-mentioned probe groups being made up of contact probe, three kinds of probes of amplification probe and fluorescence probe.
Further, the hybridization solution includes：1~10% (w/v) dextran sulfate, 100~1000mM NaCl, 10~ 40% (v/v) deionized formamide, 5mM Na2EDTA, 0.01~1% (v/v) TritonX-100,5~200mM Tris-HCl (pH 7.5), 0.1~1% Tween 80,0.1~1% betaine.
The present invention finally provides a kind of method that mentioned reagent box quick detection ALK gene is broken, and comprises the following steps：
(1) 10 μ L sample drops are drawn on slide, is dried at 56 DEG C, sample is fixed on slide；
(2) slide is immersed in dimethylbenzene at room temperature and dewaxed 2 times, each 10min then soaks in 100% ethanol 5min is steeped, each 2min rehydrations in 100% ethanol, 85% ethanol and 70% ethanol are then immersed successively, purifying is immersed at 90 DEG C 20-30min is boiled in water, slide is taken out into observation once per 5min, prevents sample from coming off, slide is taken out, at room temperature will It immerses each 2min dehydrations in 70% ethanol, 85% ethanol and 100% ethanol successively, dries naturally；
(3) 0.5 μ L contact probe 1, contact probe 2, amplification probe 1, amplification probe 2, the and of fluorescence probe 1 is drawn respectively Fluorescence probe 2, is added in 17 μ L hybridization buffers, mixes, and centrifugation obtains probe mixture；
(4) 20 μ L probe mixtures are dripped in the hybridising region of slide, cover plate, glue edge sealing, be placed in miscellaneous in hybridization instrument Hand over 30min；
(5) the mounting glue and cover glass on slide are removed, 2h is dried at 67 ± 1 DEG C, is then immersed in containing 0.3% (v/v) 2min is rinsed in NP-40 0.4 × SSC solution, rocks 1~2 time, dry naturally in the dark therebetween；
(6) 10 μ L DAPI counterstains are dripped in the hybridising region of slide, is capped cover glass, stood 10min, use fluorescence Sediments microscope inspection.
Technical key point or principle：FISH (Flourescence in situ Hybridization, FISH) be the probe that a kind of application is marked with fluorescent material, detected by the method for hybridization cell or The method for organizing internal specific DNA or RNA；Traditional FISH is limited due to the fluorescence intensity of wall scroll probe, can only Fluorescence signal intensity is improved by using the coefficient method of a plurality of probe, these probe wall scrolls length about 200~500bp, Covering amounts to the genome area for being up to more than 100Kb.Because number of probes is more, wide coverage inevitably has part Probe can produce non-specific hybridization, so as to cause background to rise.Simultaneously as the coverage of probe is excessive, many times Fluorescence signal can not accurately be polymerized to a point, but can diverge and, influence detection and result interpretation, in addition, tradition FISH Detection need to undergo the hybridization up to 16 hours, and detection time is long, and detection efficiency is low.
The present invention by introducing contact probe, three kinds of probes of amplification probe and fluorescence probe, and by these three probes with The interaction of target gene (ALK), 400 times are amplified by fluorescence signal, and hybridization time only needs 30 minutes；The present invention by Non-existent base in two kinds of n DNAs of F (C methylated) and J (G methylated) is introduced into three kinds of probes, to improve three kinds The precision of probe interaction, is effectively prevented from non-specific hybridization.
The present invention is by many experiments, and the optimum temperature for determining FISH is 42-58 DEG C, formamide optium concentration For 10-40%, the optium concentration of molecular beacon is 10ng/L.
The fluorophor that fluorescence probe 5 ' of the present invention is held, including but not limited to Cy3, FITC or FAM etc., can be according to existing Technology is added.
Compared with conventional fluorescent in-situ hybridization method, the method that the present invention is provided has advantages below：
1st, probe groups of the invention specifically recognize ALK gene broken site N, C two ends 30bp sequence, only traditional The one thousandth of method, because probe overlay area is small, the probability for producing non-specific amplification is substantially reduced, so that background is more dry Only；
2nd, probe groups length of the invention only has the one thousandth of traditional FISH methods, because probe overlay area is small, production Raw fluorescence signal is more concentrated, simultaneously as fluorescence signal is exaggerated 400 times, fluorescence signal is more easy to know than conventional method Other and detection；
3rd, the interaction DNA section of probe groups of the invention is in below 30bp, it is only necessary to just can complete miscellaneous within 30 minutes Hand over, the hybridization time than traditional FISH methods (16 hours) is shortened dramatically.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing There is the accompanying drawing used required in technology description to be briefly described, it should be apparent that, drawings in the following description are only this Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can be with Other accompanying drawings are obtained according to these accompanying drawings.
Fig. 1 is the fluorogram being broken using probe in detecting ALK gene, and wherein a is using traditional FISH probe detection ALK bases Because of the negative findings of fracture, b is the negative findings being broken using the probe groups detection ALK gene of the present invention, and c is using tradition The positive findings of FISH probe detection ALK gene fracture, d is the positive being broken using the probe groups detection ALK gene of the present invention As a result.
Embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but the following example is merely to illustrate The present invention, and should not be taken as limiting the scope of the invention.Unreceipted actual conditions person in embodiment, according to normal condition or manufacture The condition of business's suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, are that can be produced by the routine of acquisition purchased in market Product.
Embodiment 1：The design and synthesis of contact probe, three kinds of probes of amplification probe and fluorescence probe
(1) by sequence alignment and Level Structure Analysis, the ALK gene broken site each 30bp in two ends sequence difference is chosen As detection target site 1 and detection target site 2, its base sequence is respectively：
Target site 1：5’-TGCCTTGGTAAGCCGTTACCTTACCAACTA-3’
Target site 2：5’-CAGCGTCAGTTACAGACCAGAAAGTCGCCT-3’
(2) according to target site sequence, complementary probe sequence is designed, and in the repetition sequence of its 3 ' end addition, 20 group of 20 base Row, constitute contact probe 1 and contact probe 2, and its base sequence is respectively：
Contact probe 1：
5’-TAGTTGGTAAGGTAACGGCTTACCAAGGCA(GTATJCGCJCTGFTATJCCG)20-3’
Contact probe 2：
5’-AGGCGACTTTCTGGTCTGTAACTGACGCTG(AGTFAJCGCFGTAFCAAJTJ)20-3’
Repetitive sequence is compared through DNA sequence dna, and any section is mismatched on human genome, will not be produced non-specific miscellaneous Hand over, and by introducing non-natural base F and J, the possibility that further reduction non-specific signals are produced；
(3) according to the repetitive sequence of respective contact probe, corresponding amplification probe 1 and amplification probe 2, its base sequence are designed Row are respectively：
Amplification probe 1：
5’-CGGFATAJCAGFGCGFATAC(TCFACGJCFCTAJGGAFAAFG)20-3’
Amplification probe 2：
5’-FAFTTGJTACJGCGFTJACT(GCAFJTTACCGFAJJTACFT)20-3’
3 ' repetitive sequences of the end with 20 group of 20 base of amplification probe, through being compared through DNA sequence dna, and human genome Upper any section is mismatched, and will not produce non-specific hybridization, and by introducing non-natural base F and J, further reduction The possibility that non-specific signals are produced；
(4) corresponding fluorescence probe 1 and fluorescence probe 2 are designed, its base sequence is respectively：
Fluorescence probe 1：5’-CY3-JTTJTCCFTAGJGFCGTJGA-3’
Fluorescence probe 2：5’-FITC-AJGTAFFTJCGGTAAFJTGC-3’
In the present embodiment, 5 ' ends of the fluorescence probe 1 and the fluorescence probe 2 are marked with fluorophor.It is described glimmering Light group includes but is not limited to FITC, FAM or Cy3 etc., can be added according to prior art.
(5) engineer synthesizes corresponding molecular beacon
In the present embodiment, by doing thermal denaturation curve experiment to molecular beacon, the optimal temperature of FISH is determined Spend for 42-58 DEG C, formamide optium concentration is 10-40%, molecular beacon optium concentration is 10ng/L.
Embodiment 2：The probe groups detection ALK gene fracture of traditional FISH probe and the present invention
A, traditional FISH probe detection
The method that ALK gene is broken is detected using traditional FISH probe, is comprised the following steps：
(1) 10 μ L sample drops are drawn on slide, is dried at 56 DEG C, sample is fixed on slide；
(2) slide is immersed in dimethylbenzene at room temperature and dewaxed 2 times, each 10min then soaks in 100% ethanol 5min is steeped, each 2min rehydrations in 100% ethanol, 85% ethanol and 70% ethanol are then immersed successively, purifying is immersed at 90 DEG C 20-30min is boiled in water, slide is taken out into observation once per 5min, prevents sample from coming off, slide is taken out, at room temperature will It immerses each 2min dehydrations in 70% ethanol, 85% ethanol and 100% ethanol successively, dries naturally；
(3) 3 μ L probes are drawn to be added in 7 μ L hybridization buffers, flicks of short duration centrifugation after centrifugation bottom of the tube is mixed, obtain Probe mixture；
(4) 10 μ L probe mixtures are dripped in the hybridising region of slide, slide is capped immediately, mounting glue seals cover glass Edge, it is to avoid bubble is produced between cover glass and slide；
(5) slide is placed in hybridization instrument, is 83 DEG C in denaturation temperature, denaturation time is 5min, and hybridization temperature is 42 Hybridize 16h under conditions of DEG C；
(6) the mounting glue and cover glass on slide are removed, 2h is dried at 67 ± 1 DEG C, is then immersed in containing 0.3% (v/v) 2min is rinsed in NP-40 0.4 × SSC solution, slide is rocked therebetween 1~2 time；
(7) slide is immersed in 2 × SSC solution containing 0.1% (v/v) NP-40 at room temperature and rinses 30s, shaken therebetween Dynamic load slide 1~2 time；
(8) slide is immersed to each 2min dehydrations in 70% ethanol, 85% ethanol and 100% ethanol successively at room temperature, Then dry naturally in the dark；
(9) 10 μ L DAPI counterstains are added dropwise in the hybridising region of slide, cover glass is capped, it is to avoid cover glass is with carrying Bubble is produced between slide.10 minutes are stood, fluorescence microscopy is used.
B, the present invention probe in detecting
The method being broken using the probe groups detection ALK gene of the present invention, is comprised the following steps：
(1) 10 μ L sample drops are drawn on slide, is dried at 56 DEG C, sample is fixed on slide；
(2) slide is immersed in dimethylbenzene at room temperature and dewaxed 2 times, each 10min then soaks in 100% ethanol 5min is steeped, each 2min rehydrations in 100% ethanol, 85% ethanol and 70% ethanol are then immersed successively, purifying is immersed at 90 DEG C 20-30min is boiled in water, slide is taken out into observation once per 5min, prevents sample from coming off, slide is taken out, at room temperature will It immerses each 2min dehydrations in 70% ethanol, 85% ethanol and 100% ethanol successively, dries naturally；
(3) 0.5 μ L contact probe 1, contact probe 2, amplification probe 1, amplification probe 2, the and of fluorescence probe 1 is drawn respectively Fluorescence probe 2, is added in 17 μ L hybridization buffers, mixes, and centrifugation obtains probe mixture；
(4) 20 μ L probe mixtures are dripped in the hybridising region of slide, slide is capped immediately, mounting glue seals cover glass Edge, it is to avoid bubble is produced between cover glass and slide；
(5) slide is placed in hybridization instrument, is 83 DEG C in denaturation temperature, denaturation time is 5min, and hybridization temperature is 42 Hybridize 30min under conditions of DEG C；
(6) the mounting glue and cover glass on slide are removed, 2h is dried at 67 ± 1 DEG C, is then immersed in containing 0.3% (v/v) 2min is rinsed in NP-40 0.4 × SSC solution, rocked therebetween 1~2 time, and dry naturally in the dark；
(7) 10 μ L DAPI counterstains are added dropwise in the hybridising region of slide, cover glass is capped, it is to avoid cover glass is with carrying Bubble is produced between slide, 10 minutes is stood, uses fluorescence microscopy.If the sample is normal cell, it can be seen that two Yellow signal, or red closely, green florescent signal, can if is the cell of ALK gene fracture occurs for the sample There are the red spread out, green florescent signal.
(8) calculate that each gene is red, green fluorescence intensity ratio (Ratio values)：Randomly select observation in fluorescence microscope 30 cells, the total and green signal of red signal counted in the nucleus of 30 cells is total, calculates according to the following formula：
Green signal sum in red signal sum/30 nucleus in the nucleus of Ratio value=30.
(9) testing result is judged according to the Ratio values of calculating：
If Ratio values<2, then the sample is negative findings, illustrates that the sample is broken without ALK gene；If Ratio values>2, then The sample is positive findings, illustrates that the sample has ALK gene fracture.
C, two methods interpretation of result
Through examining, the FISH methods that the present invention is provided are compared with traditional FISH methods, and what sample yin and yang attribute was judged is consistent Property more than 99.9%, but tradition FISH methods need hybridization 16 hours, FISH methods of the invention need to only hybridize 30 minutes.And this The FISH method backgrounds of invention are cleaner, and fluorescence signal is apparent, as shown in Figure 1.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected The limitation of scope is protected, although being explained in detail with reference to preferred embodiment to the present invention, one of ordinary skill in the art should Understand, technical scheme can be modified or equivalent substitution, without departing from the essence of technical solution of the present invention And scope.

Claims (9)

1. a kind of probe groups of quick detection ALK gene fracture, it is characterised in that visited by contact probe, amplification probe and fluorescence Three kinds of probe compositions of pin, the contact probe includes contact probe 1 and contact probe 2, and the amplification probe includes amplification probe 1 With amplification probe 2, the fluorescence probe includes fluorescence probe 1 and fluorescence probe 2, wherein, it is contact probe 1, amplification probe 1, glimmering Light probe 1, contact probe 2, the base sequence of amplification probe 2 and fluorescence probe 2 are respectively：
Contact probe 1：
5’-TAGTTGGTAAGGTAACGGCTTACCAAGGCA(GTATJCGCJCTGFTATJCCG)-3’；
Amplification probe 1：
5’-CGGFATAJCAGFGCGFATAC(TCFACGJCFCTAJGGAFAAFG)-3’；
Fluorescence probe 1：5’-CY3-JTTJTCCFTAGJGFCGTJGA-3’；
Contact probe 2：
5’-AGGCGACTTTCTGGTCTGTAACTGACGCTG(AGTFAJCGCFGTAFCAAJTJ)-3’；
Amplification probe 2：
5’-FAFTTGJTACJGCGFTJACT(GCAFJTTACCGFAJJTACFT)-3’；
Fluorescence probe 2：5’-FITC-AJGTAFFTJCGGTAAFJTGC-3’.
2. probe groups according to claim 1, it is characterised in that 5 ' ends of the fluorescence probe 1 and the fluorescence probe 2 Marked with fluorophor.
3. probe groups according to claim 2, it is characterised in that the fluorophor is CY3 or FITC.
4. probe groups according to claim 1, it is characterised in that the contact probe 1, the amplification probe 1 and described Fluorescence probe 1 is used to detect ALK gene broken site N-terminal, and the fluorescence probe 1 is by ALK gene broken site N-terminal labeled as red Color fluorescence, the contact probe 2, the amplification probe 2 and the fluorescence probe 2 are used to detect ALK gene broken site C-terminal, ALK gene broken site N-terminal is labeled as green fluorescence by the fluorescence probe 2.
5. probe groups according to claim 4, it is characterised in that the contact probe, the amplification probe and described glimmering F and J in the base sequence of light probe are non-existent base in two kinds of natural gene group DNA, wherein, F represents what is methylated C, J represent the G methylated.
6. a kind of be used for the kit that quick detection ALK gene is broken containing any probe groups of claim 1-5.
7. kit according to claim 6, it is characterised in that also including including hybridization solution.
8. kit according to claim 7, it is characterised in that the hybridization solution includes：1~10% (w/v) sulfuric acid Portugal Glycan, 100~1000mM NaCl, 10~40% (v/v) deionized formamides, 5mM Na2EDTA, 0.01~1% (v/v) In TritonX-100,5~200mM Tris-HCl (pH 7.5), 0.1~1% Tween 80,0.1~1% trimethylammonium second Ester.
9. the method that kit quick detection ALK gene described in a kind of utilization claim 6 is broken, it is characterised in that including such as Lower step：
(1) 10 μ L sample drops are drawn on slide, is dried at 56 DEG C, sample is fixed on slide；
(2) slide is immersed in dimethylbenzene at room temperature and dewaxed 2 times, each 10min then soaks in 100% ethanol 5min, then immerses each 2min rehydrations in 100% ethanol, 85% ethanol and 70% ethanol, purified water is immersed at 90 DEG C successively In boil 20-30min, per 5min by slide take out observation once, prevent sample from coming off, take out slide, at room temperature by it Each 2min dehydrations in 70% ethanol, 85% ethanol and 100% ethanol are immersed successively, are dried naturally；
(3) 0.5 μ L contact probe 1, contact probe 2, amplification probe 1, amplification probe 2, fluorescence probe 1 and fluorescence is drawn respectively Probe 2, is added in 17 μ L hybridization buffers, mixes, and centrifugation obtains probe mixture；
(4) 20 μ L probe mixtures are dripped in the hybridising region of slide, cover plate, glue edge sealing, is placed in hybridization instrument and hybridizes 30min；
(5) the mounting glue and cover glass on slide are removed, 2h is dried at 67 ± 1 DEG C, is then immersed in containing 0.3% (v/ V) 2min is rinsed in NP-40 0.4 × SSC solution, rocks 1~2 time, dry naturally in the dark therebetween；
(6) 10 μ L DAPI counterstains are dripped in the hybridising region of slide, is capped cover glass, stood 10min, use fluorescence microscopy Mirror microscopy.
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Citations (8)

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