CN203360442U - 用于结核病快速诊断及药效检测的基因数组结构 - Google Patents
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Abstract
一种用于结核病快速诊断及药效检测的基因数组结构,包括基板、结核杆菌检测区以及抗药性检测区。该基板上具有第一、二检测框及数个样本滴孔,该两检测框内的样本滴孔分别承接结核杆菌中特异性区段样本与抗结核药物样本;该结核杆菌检测区具有数个包含结核杆菌特异性基因的基因检测点;该抗药性检测区具有数个包含抗药性基因的基因检测点。如此,以结核杆菌特异基因与抗药性基因为探针,以杂合反应进行分析,可同时检测结核杆菌与抗药性基因,达到快速、低成本并能兼测抗结核药物的抗药性等功效。
Description
技术领域
本实用新型有关于一种结核病快速诊断及药效检测结构,特别指兼具结核杆菌分型与抗药性分析,可同时检测结核杆菌与抗药性基因的基因数组结构。
背景技术
结核病(Tuberculosis, TB)是一种相当古老的传染性疾病,虽然全球科学家经过长久的努力,研究预防、控制及治疗的方法,然而,结核病目前仍然是全球重要的公共卫生上的问题,其同时也是单一病原引起死亡病例最高的传染病。根据世界卫生组织(World Health Organization, WHO)的预估,全球超过三分之一的人口,已经受过结核分枝杆菌(Mycobacterium Tuberculosis)的感染,经统计显示,每年大约新增八百万结核病新病例且造成两百万人因结核病而死亡。
结核病的特色在于受到结核分枝杆菌感染后,通常并不会立即发病,感染者仅约10%的机率发病而成为活动性结核病患者(Active TB)。另大部分患者受结核分枝杆菌感染后,菌体可长期潜存在宿主体内等待伺机发病。当结核分枝杆菌及宿主防御机转之间微妙的平衡破坏,潜伏性病灶才会活化而成为活动性患者。 因此,整体而言,潜藏性结核病患(Latent TB)转变为活动性结核病患的发病成因,可能是再次感染(Exogenous Reinfection),也有可能是病原菌的再活化(Endogenous Reactivation)。
结核病的临床表现千变万化,发病之初往往没有明显或特异性的症状,且病程发展缓慢,时好时坏,临床诊断上十分困难。因此,临床上结核病的诊断,必须综合患者的临床表征,加上X 光片上的变化,最后再以实验室检验加以证实才能正确判断。
在结核病的实验诊断方面,主要以组织病理学、抗酸菌染色与肺结核杆菌培养等技术为主。然而这些检验技术都有它们的检测局限。以痰液抹片的显微镜染色检查而言,其是传统方式中最快检测抗酸菌的方法,但痰液检体中,必需每毫升(ml)至少含5000至10000只菌,才可由染色的抹片镜检查出。另外,此技术的伪阳性也很高,因为除了结核分枝杆菌外,非典型分枝杆菌(Non-Tuberculous Mycobacteria, NTM)及少数其它的杆菌,亦可呈染色阳性。至于结核分枝杆菌的培养方法为传统上结核病的确定诊断方法之一,虽有敏感性为80至85%,特异性为98%的特性,惟其需要至少4到8周的时间才能得到检验结果,时效上严重达不到临床上的需求。
随着分子生物技术的蓬勃发展,且广泛应用于生物医学的探讨,利用分子生物学相关技术,如聚合酶连锁反应(Polymerase Chain Reaction, PCR)与聚合酶连锁反应-限制酵素切割片段长度多型性(PCR-Restriction Fragment Length Polymorphism, PCR-RFLP)等,对结核病进行分子诊断,使结核病的临床诊断有了革命性的进步。发展初期,一般检验单位多利用聚合酶连锁反应,直接侦测受检者痰液检体之中是否存在肺结核分枝杆菌的分子标记,如热休克蛋白65(hsp65)与插入片段6110(IS6110)的脱氧核醣核酸(Deoxyribonucleic Acid, DNA)或核醣核酸(Ribonucleic Acid, RNA)。并结合限制酵素切割片段长度多型性,对所侦测的菌体核酸进行分子分型。然而,此种方法虽可快速地侦测结核杆菌的存在,但不管检测宿主检体中所含结核分枝杆菌特有的DNA或讯息核醣核酸(messenger RNA, mRNA)表现量,其在临床诊断上仍旧无法达到令人满意的正确性。
这几年,由于结核分枝杆菌全基因体的译码,科学家们发现结核分枝杆菌与牛分枝杆菌减毒菌株(Mycobacterium bovis BCG)之间存在基因体缺失(Genomic Deletion)的现象,并将缺失片段位置统称为差异性区段(Regions-of-Difference, RD)。许多研究报告指出,此种差异性区段造成的原因可能为基因体中DNA复制时的错误或是插入性片段的插入,造成基因体的片段缺失(Deletion)、插入(Insertion)、倒置(Inversion)或复制(Replication)等,最后终于造成菌种间差异性区段的产生。
差异性区段上存有许多重要的基因以及致病因子,这些区段的存在或缺失可因而造成结核杆菌群中各个菌种致病性的不同。因此,以差异性区段作为结核分枝杆菌分型标的,不但可保有分子诊断技术快速敏感性高的优点,亦可弥补传统分子检测方式无法鉴定结核分枝杆菌群中各菌株的缺点,是相当具潜力性结核病诊断的分子标记。
黄等人曾于2009年发表在每一个差异性区段中选出特异性目标基因共14个,作为建构结核病基因诊断芯片的检测标的。并利用可同时检测多基因标的的微量核酸检测技术平台,使其灵敏度可达每毫升全血中只要有5个细胞即可检测,藉此建立以痰液检体为检测对象的结核病基因诊断芯片。
该实验先自临床检体分离致病性分枝杆菌群菌株,以传统生化反应及核酸定序确认分离菌株后,记录每一病患所感染的结核菌作为标准参考数据,继而利用现行分子检测技术PCR-RFLP及基因芯片检测技术,直接由结核病患者的痰液检体侦测结核分枝杆菌的存在,并且进一步比较两种方法间的一致性及方便性。结果显示,由收集的246个结核病患者的临床痰液检体中,可直接以PCR-RFLP侦测出结核分枝杆菌群(TB Complex, TBC)存在的比例为62.5%,而以基因芯片检测技术可侦测出的比例为85%;并且,此两种方法检测出的菌株种类完全一致。分析两种方法检测结果与痰液染色的相关性,在56个阳性培养且痰液染色阳性的检体中,PCR-RFLP可侦测出39个,而基因芯片检测技术的检出率可大于90%,能侦测出52个;而在24个阳性培养但痰液染色呈阴性的检体中,PCR-RFLP可检测出11个,而基因芯片检测技术的检出率可高达67%,能侦测出16个。
由上述结果可知基因芯片检测技术不但操作方便,可有效节省人力、时间的耗费,其灵敏度更远比PCR-RFLP技术高出许多,是结核病分子诊断未来发展的新趋势。
然而,针对世界市面上结核分枝杆菌检测/药效检测套组,如Spoligotyping Method(荷兰-Isogen)、TB Ag Rapid Test(中国台湾-台塑生医科技)、Amplified MTDR(美国-Gene Probe)、DR. MTBC Screen Kit(中国台湾-晶宇生物科技)及GenoType MTBDRplus(德国-Hain Lifescience)。其中Spoligotyping Method系检测结核分枝杆菌特异性间隔寡核苷酸图谱,再从已建立的数据库进行比对分型,惟其分辨菌株能力不及标准方法,具有鉴别率低的缺点,并且不具抗药性分析的药效检测功能;TB Ag Rapid Test系以结核杆菌特异性抗原,利用抗原抗体反应直接侦测结核菌,惟其具有成本高,并需进行分枝杆菌菌落培养,步骤繁杂且耗时的缺点,同时亦不具抗药性分析的药效检测功能;Amplified MTDR系检测结核杆菌核醣体核醣核酸(ribosomal RNA, rRNA)与特异性DNA探针杂合并以冷光侦测,惟其需进行分枝杆菌菌落培养,具有步骤繁杂且耗时的缺点,并同样不具抗药性分析的药效检测功能;DR. MTBC Screen Kit系针对结核杆菌内特有之基因片段,以PCR进行放大,再利用芯片进行探针杂合反应,虽可采以痰液样本进行结核杆菌检测并找出结核杆菌群的Rifampicin的抗药性基因,惟其每毫升的痰中需有10万只细菌才能验出,敏感度也仅有65%,具有敏感度低的缺点;GenoType MTBDRplus是以PCR增幅结核杆菌抗药性基因,再与探针进行杂合反应,虽可找出结核杆菌群的Ofloxacin、Streptomycin及Ethambutol的抗药性基因,惟其具有成本高,技术复杂度高、耗时且需持续品管的缺点,并且仅具药效检测而不具结核杆菌检测功能。
另外,按中国台湾申请专利第201005098号的活动性肺结核杆菌芯片检测法,其是一种能直接经宿主痰液检体检测结核杆菌感染的活动期肺结核杆菌诊断芯片,惟其仅能提供结核菌筛检,并不具药效分析基因群的功能。故,一般无法符合使用者于实际使用时达到快速诊断结核病的同时兼具药效检测抗药性的所需。
发明内容
本实用新型的目的是,克服已知技术所遭遇的上述问题并提供一种兼具结核杆菌分型与抗药性分析,可用于结核病快速诊断及药效检测的基因数组结构。
为了达到上述目的,本实用新型所采用的技术方案是:一种用于结核病快速诊断及药效检测的基因数组结构,其特点是,其包括:基板,其上具有第一检测框、第二检测框、及数个可供容纳各种基因检测片段的样本滴孔,且该样本滴孔于该基板表面按照行与列整齐地呈数组状排列,其中该第一检测框内的样本滴孔承接结核杆菌中特异性区段样本,该第二检测框内的样本滴孔则供承接抗结核药物样本;结核杆菌检测区,具有数个被覆于该基板表面且分别接合有可与特定生物分子反应而产生不同呈色表现的生物探针的基因检测点于该第一检测框内的样本滴孔中,该些基因检测点中包含13个结核杆菌特异性基因的结核杆菌特异性基因群,且该13个结核杆菌特异性基因分别为hsp65、Rv0577、Rv3120、Rv2073c、Rv1970、Rv3875、Rv3347c、Rv1510、Rv0186、Rv0124、TbD1、mtp40及mpb83;以及抗药性检测区,其具有数个被覆于该基板表面且分别接合有可与Isoniazid、Rifampicin、Ofloxacin、Ethambutol及Streptomycin的抗结核药物反应而产生不同呈色表现的基因检测点于该第二检测框内的样本滴孔中,该些基因检测点中包含6个抗药性基因的抗药性基因群,且该6个抗药性基因分别为katG、rpoB、gyrA、embB、rpsL及rrs。
所述基因检测点呈数组排列状。
所述结构更包括阳性控制组、阴性控制组及空白控制组。
如此,以结核杆菌特异基因与抗药性基因为探针,以杂合反应进行分析,可同时检测结核杆菌与抗药性基因,达到快速、低成本并能兼测抗结核药物的抗药性等功效。
附图说明
图1是本实用新型基因数组结构的检测区块示意图。
图2是本实用新型基因数组结构之基因排列示意图。
图3是本实用新型一较佳实施例的结核杆菌检测区判读示意图。
图4是本实用新型一较佳实施例的抗药性检测区判读示意图。
标号说明
基因数组结构20 结核杆菌检测区21
抗药性检测区22 基板23
第一检测框231 第二检测框232
样本滴孔233 基因检测点2a、2b。
具体实施方式
请参阅图1及图2所示,分别为本实用新型基因数组结构的检测区块示意图及本实用新型基因数组结构的基因排列示意图。如图所示:本实用新型为一种用于结核病快速诊断及药效检测的基因数组结构,至少包括由基板23、结核杆菌检测区21及抗药性检测区22所构成的基因数组结构20,图中P代表阳性控制组、N代表阴性控制组以及B代表空白控制组。
上述所提的基板23,其上具有第一检测框231、第二检测框232、及数个可供容纳各种基因检测片段的样本滴孔233,且该等样本滴孔233于该基板23表面按照行与列整齐地呈数组状排列,其中该第一检测框231内的样本滴孔233为供承接结核杆菌中特异性区段样本,该第二检测框232内的样本滴孔233则供承接抗结核药物样本。
上述所提的结核杆菌检测区21,具有数个被覆于该基板23表面且分别接合有可与特定生物分子反应而产生不同呈色表现的生物探针的基因检测点2a,该第一检测框231内的样本滴孔233中,该些基因检测点2a中包含13个结核杆菌特异性基因的结核杆菌特异性基因群,其中,具诊断结核杆菌的结核杆菌特异性基因群,主要包含如表1所示的结核杆菌特异性基因所挑选的特异性寡核苷酸序列,而由表1可知此包含13个结核杆菌特异性基因的结核杆菌特异性基因群分别为hsp65、Rv0577、Rv3120、Rv2073c、Rv1970、Rv3875、Rv3347c、Rv1510、Rv0186、Rv0124、TbD1、mtp40及mpb83。
表1结核杆菌特异性基因群
上述所提的抗药性检测区22,具有数个被覆于该基板23表面且分别接合有可与Isoniazid、Rifampicin、Ofloxacin、Ethambutol及Streptomycin等抗结核药物反应而产生不同呈色表现的基因检测点2b于该第二检测框232内的样本滴孔233中,该些基因检测点2b中包含6个抗药性基因的抗药性基因群,其中,具检测抗结核药物药效的抗药性基因群,主要包含如表2所示的抗药性基因所挑选的序列,而由表2可知此包含6个抗药性基因的抗药性基因群分别为katG、rpoB、gyrA、embB、rpsL及rrs。
表2抗药性基因群
请参阅图3所示,为本实用新型的一较佳实施例的结核杆菌检测区判读示意图。如图所示:本实用新型将13个选定的结核杆菌特异性基因以及6个选定的抗药性基因,于一尼龙膜片上建构基因数组结构,其中该结核杆菌检测区21经一较佳实施例,以结核杆菌特异基因为探针,由杂合反应进行分析,再以呈色反应检测杂合讯息,若有呈色反应即代表有该基因表现,并依照此基因表现差异进行结核杆菌分型。图中显示:结核杆菌于该基因数组结构呈色结果为hsp65(+)、Rv0577(+)、Rv3120(-)、Rv2073c(-)、TbD1(+)、Rv1970(-)、Rv3875(+)、Rv3347c(+)、Rv1510(-)、Rv0186(+)、Rv0124(+)、mtp40(+)以及mpb83(+);其中判读结果为(+)者代表阳性反应。
更详细的比较可由下表3所示。
表3
请参阅图4所示,本实用新型一较佳实施例的抗药性检测区判读示意图。如图所示:本实用新型将13个选定的结核杆菌特异性基因以及6个选定的抗药性基因,于一尼龙膜片上建构基因数组结构,其中以该抗药性检测区22中Ethambutol药效检测为例,并以embB-W1设定为一定会呈色出现的embB阳性控制组,而embB-W306、embB-W319与embB-W406为野生型探针embB codon 306、319与406,且以embB-Q306、embB-Q319与embB-Q406作为容易突变的位点所带有之内控制组embB codon 306、319与406。其经一较佳实施例,以抗药性基因为探针,由杂合反应进行分析,再以呈色反应检测杂合讯息以分析抗药性基因是否有突变,若有突变则无法与野生型探针接上,进而无法呈色。图中显示:抗药性基因embB于该基因数组结构呈色结果为codon 306(-)、codon 319(+)以及codon 306(+);其判读结果为embB codon 306突变,显示此菌对于Ethambutol有抗药性。
综上所述,本实用新型为一种用于结核病快速诊断及药效检测的基因数组结构,可有效改善现有技术的种种缺点,是以结核杆菌特异基因与抗药性基因为探针,以杂合反应进行分析,可同时检测结核杆菌与抗药性基因,达到快速、低成本并能兼测抗结核药物的抗药性等功效,进而能产生更进步、更实用、更符合使用者所须,确已符合新型专利申请的要件,依法提出专利申请。
惟以上所述,仅为本实用新型的较佳实施例而已,当不能以此限定本实用新型实施的范围;故,凡依本实用新型申请专利范围及新型说明书内容所作的简单的等效变化与修饰,皆应仍属本实用新型专利涵盖的范围内。
Claims (3)
1.一种用于结核病快速诊断及药效检测的基因数组结构,其特征在于,其包括:
基板,其上具有第一检测框、第二检测框、及数个可供容纳各种基因检测片段的样本滴孔,且该样本滴孔于该基板表面按照行与列整齐地呈数组状排列,其中该第一检测框内的样本滴孔承接结核杆菌中特异性区段样本,该第二检测框内的样本滴孔则供承接抗结核药物样本;
结核杆菌检测区,具有数个被覆于该基板表面且分别接合有可与特定生物分子反应而产生不同呈色表现的生物探针的基因检测点于该第一检测框内的样本滴孔中,该些基因检测点中包含13个结核杆菌特异性基因的结核杆菌特异性基因群,且该13个结核杆菌特异性基因分别为hsp65、Rv0577、Rv3120、Rv2073c、Rv1970、Rv3875、Rv3347c、Rv1510、Rv0186、Rv0124、TbD1、mtp40及mpb83;以及
抗药性检测区,其具有数个被覆于该基板表面且分别接合有可与Isoniazid、Rifampicin、Ofloxacin、Ethambutol及Streptomycin的抗结核药物反应而产生不同呈色表现的基因检测点于该第二检测框内的样本滴孔中,该些基因检测点中包含6个抗药性基因的抗药性基因群,且该6个抗药性基因分别为katG、rpoB、gyrA、embB、rpsL及rrs。
2.如权利要求1所述的用于结核病快速诊断及药效检测的基因数组结构,其特征在于,所述基因检测点呈数组排列状。
3.如权利要求1所述的用于结核病快速诊断及药效检测的基因数组结构,其特征在于,所述结构更包括阳性控制组、阴性控制组及空白控制组。
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CN106093420A (zh) * | 2016-05-30 | 2016-11-09 | 南华大学 | 一种用于结核病血清学诊断的elisa试剂盒 |
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CN106093420A (zh) * | 2016-05-30 | 2016-11-09 | 南华大学 | 一种用于结核病血清学诊断的elisa试剂盒 |
CN106093420B (zh) * | 2016-05-30 | 2017-09-29 | 南华大学 | 一种用于结核病血清学诊断的elisa试剂盒 |
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