FQ-PCR检测不孕不育患者解脲支原体和沙眼衣原体

目的观察解脲支原体和沙眼衣原体的感染与不孕不育的关系。探讨实时荧光定量PCR技术在不孕不育患者的诊断和治疗中的作用。方法采用实时荧光定量PCR技术检测45对不孕不育夫妇女性宫颈分泌物、男性精液或前列腺标本中的解脲支原体-DNA和沙眼衣原体-DNA含量。结果不孕不育患者解脲支原体-DNA阳性率37.78%,沙眼衣原体-DNA阳性率28.89%,解脲支原体或沙眼衣原体的总感染率为52.22%,正常对照组则分别为15.00%、8.33%、20.00%,两组间有显著性差异。不孕不育组中解脲支原体的感染率明显高于沙眼衣原体（P〈0.05）。女性患者解脲支原体DNA载量和沙眼衣原体-DNA载量均高于男性患者（P〈0.05）。结论解脲支原体和沙眼衣原体是引起男女不孕不育的重要原因之一。荧光定量PCR技术用于诊断引起不孕不育的解脲支原体或沙眼衣原体泌尿生殖道感染是完全可靠的。     

单核细胞乙型肝炎病毒cccDNA检测方法及应用价值

目的 建立检测HBV共价闭合环状DNA（ cccDNA）的巢式-荧光定量PCR法,检测外周血单核细胞（ PBMC）及骨髓单核细胞（MMNC）中cccDNA。方法 根据HBV cccDNA与松弛结构DNA（rcDNA）结构上的差异,设计2对跨缺口的特异性引物及下游的特异性TaqMan探针。根据Mung Bean Nuclease对rcDNA与cccDNA作用的不同,使cccDNA扩增而使rcDNA降解,分别用外引物及内引物进行PCR反应,再用荧光探针进行实时荧光定量PCR,根据阳性参照物,计算出检测标本定量值。结果 成功建立了HBV cccDNA巢式-荧光定量PCR的检测方法,线性范围为5.0× 10２～3.9×107拷贝/ml。用上述方法检测25例慢乙肝及肝硬化血清HBV DNA阳性患者外周血单核细胞中cccDNA,7例骨髓单核细胞中cccDNA,21例健康献血者外周血单核细胞中cccDNA,骨髓标本中有3例cccDNA阳性,25例外周血标本中有9例cccDNA阳性。结论 巢式-荧光定量PCR法可检测乙肝患者PBMC及MMNC中的HBVcccDNA含量。PBMC及MMNC中可检测到HBVcccDNA.     

荧光定量PCR检测沙眼衣原体及临床应用

目的：用荧光定量聚合酶链反应（FQ－PCR）检测沙眼衣原体的含量,探讨FQ－PCR在衣原体性尿道炎诊断中的价值。方法：应用荧光探针标记引物的荧光定量聚合酶链反应对172例疑为沙眼衣原体感染患者标本进行检测,并与常规PCR（电泳－EB染色）进行比较。结果：FQ－PCR阳性率为19．8％,常规PCR法为20．9％,两法符合率为63．9％。女性宫颈分泌物标本FQ－PCR阳性为29．2％,常规PCR法为16．7％;男性分泌物FQ－PCR阳性率为16．9％,常规PCR法为13．8％。FQ－PCR特异性较常规PCR法高。结论;FQ－PCR在扩增中实时在线检测,并选择理想的标准曲线做出较精确的临值分析,具有较高的特异性和敏感性,对病原体的诊断有一定的临床意义。     

荧光定量PCR对不育症患者解脲支原体和沙眼衣原体感染情况的研究

目的用实时荧光定量PCR(FQ-PCR)定量检测不孕不育患者解脲支原体(UU)、沙眼衣原体(CT)基因,观察UU、CT感染情况与不孕不育的关系,探讨实时荧光定量PCR在不孕不育患者诊断和治疗中作用。方法采用FQ-PCR技术检测标本402份。结果 UU阳性率是32.8%,其中男性UU阳性率是21.4%,女性UU阳性率是44.3%。CT阳性率是16.2%,其中男性CT率是11.4%,女性CT率是20.9%。UU感染率在男性、女性患者有极显著性差异(P<0.01),而CT感染率在性别之间有显著性差异(P>0.05)。结论解脲支原体(UU)、沙眼衣原体(CT)是引起男女不育的主要原因之一,实时荧光定量PCR检测UU、CT基因具有操作简单、反应时间短、重复性好、结果客观准确、敏感性和特异性好等优点,其结果对临床诊断、治疗有一定指导意义。     

荧光定量PCR对1501例NGU患者病原体基因检测结果分析

目的用实时荧光定量聚合酶链反应（FQ-PCR）技术准确度定量检测江门市区凝有非淋菌性尿道炎患者沙眼衣原体（CT）、解脲支原体（UU）基因,进一步了解UU、CT的感染情况,为临床治疗提供依据。方法运用实时荧光定量PCR法（FQ-PCR）对1501例凝有非淋菌性尿道炎患者同时进行CT、UU检测。结果CT的阳性率是13.1%,UU的阳性率是33.9%,二者之间阳性率比较有显著性差异。结论1.江门市区非淋菌性尿道炎患者病原体CT、UU中占有一定比例,二者之间感染率比较有显著性差异。2.荧光实时定量PCR检测UU、CT具有操作简单、反应时间短、重复性好、结果客观准确、敏感性和特异性好等优点,其结果对临床诊断、治疗有一定指导意义。     

荧光定量PCR检测查菲埃立克体

依据查菲埃立克体16SrRNA基因序列设计特异性引物和TaqMan-MGB探针,以克隆的查菲埃立克体16SrRNA基因片段作DNA模板,建立实时荧光定量PCR检测方法。与套式PCR相比较,荧光定量PCR检测的灵敏度是其30倍;用荧光定量PCR检测其他相关立克次体和细菌DNA样本,检出结果为0;对荧光定量PCR检测重复性进行分析,变异系数(CV)批内和批间误差在0·2%~2·0%之间。结果证明本研究建立的荧光定量PCR方法具有种特异性和良好的重复性,可用于检测感染样本中的微量查菲埃立克体DNA。     

肝螺杆菌TaqMan MGB探针实时荧光定量PCR快速检测方法的建立及应用研究

目的 建立特异、敏感、快速检测肝螺杆菌的TaqMan MGB探针实时荧光定量PCR方法.方法 针对肝螺杆菌flaB 基因的保守区设计特异性引物和探针,建立肝螺杆菌TaqMan MGB探针实时荧光定最PCR方检测方法,验证方法的特异性、敏感性和稳定性.对2008-2011年期间采集的1081份临床样本中的肝螺杆菌进行检测,同时进行分离培养和常规PCR检测.结果 建立的TaqMan MGB探针实时荧光定量PCR方法对肝螺杆菌的检测具有高度的特异性,对幽门螺杆菌、空肠弯曲菌、泰泽氏菌、侵肺巴斯德氏菌、大肠埃希菌、铜绿假单胞菌均无交叉反应,检测的灵敏度达8.3拷贝.标准曲线显示各浓度范围内具有良好的线性关系,相关系数为0.999,斜率为-3.227,TaqManMGB探针实时荧光定量PCR效率为100％.对1081份临床样本进行检测,TaqMan MGB探针实时荧光定量PCR和常规PCR均能检出86份肝螺杆菌阳性样本,而细菌分离培养则仅检出4份阳性.结果显示,建立的TaqMan MGB探针实时荧光定量PCR方法比细菌分离培养方法更敏感,能够直接从临床样本中检出肝螺杆菌DNA,检测时间仅为2h.结论 研究建立的TaqMan MGB探针实时荧光定量PCR方法具有可靠、特异、敏感的特点,适用于肝螺杆菌的快速检测.     

不孕不育夫妇CT、UU检测结果分析及临床意义

目的了解解脲支原体（UU）和沙眼衣原体（CT）感染与不孕不育的关系,同时了解不孕不育夫妇之间的交叉感染情况,为生殖医学提供相关的医学依据,以便采取相应的治疗和有效预防措施,提高受孕率。方法用荧光定量PCR方法检测200对夫妇（不孕不育组100对,正常对照组100对）女性宫颈分泌物、男性精液标本中的解脲支原体一DNA和沙眼衣原体一DNA含量,并对相关实验数据进行医学统计学处理。结果不孕不育组女性解脲支原体-DNA阳性率46%,沙眼衣原体-DNA阳性率25%,解脲支原体或沙眼衣原体的总感染率为55%,男性解脲支原体-DNA阳性率21%,沙眼衣原体-DNA阳性率20%,解脲支原体或沙眼衣原体的总感染率为36%,一对夫妇同时感染同一种病原体者32%;正常对照组女性则分别为15%、9%、20%,男性分别为12%、6%、15%,一对夫妇同时感染同一种病原体者11%,两组间有显著性差异。结论解脲支原体和沙眼衣原体是引起男女不孕不育的重要病原体之一,应引起生殖医学工作者的足够重视,同时积极加强不孕不育夫妇之间交叉感染的预防,也是提高不孕不育夫妇受孕率的方法之一。     

荧光定量PCR检测新疆地区不孕不育患者UU、CT结果分析

目的利用实时荧光定量PCR技术了解新疆地区不孕不育患者解脲支原体（UU）和沙眼衣原体（CT）感染情况,通过对实验结果的统计学处理分析不孕不育与UU、CT感染率及感染量之间的关系,方法采用实时荧光定量PCR技术检测新疆地区不孕不育患者及能正常生育人群解脲支原体-DNA和沙眼衣原体-DNA含量并进行统计学分析。结果不孕不育患者UU-DNA阳性率52.18%,CT-DNA阳性率3.65%,正常对照组则分别为46.08%、1.00%,两组间的差异显著性有统计学上的意义,不孕不育组UU和CT的感染率明显高于对照组（P〈0.05）。不同性别,不用民族的UU阳性检出率不同。结论新疆地区不孕不育患者中UU和CT感染是引起男女不孕不育的重要原因之一,且女性UU阳性率高于男性,维吾尔族UU阳性率高于其它民族。     

双重荧光PCR检测HIV前病毒DNA及其在婴幼儿HIV感染早期诊断中的应用

目的建立双重荧光PCR检测HIV前病毒DNA的方法,并应用于婴幼儿HIV感染的早期诊断。方法采用TaqMan技术,组建针对人类核糖核酸酶P（ RNase P）和HIV的长末端重复序列（ LTR）基因的双重荧光PCR体系;采用TA克隆技术构建pTG19-T重组质粒作为模板进行该方法灵敏度的评价;采用11例已知健康人的血样和98例已知HIV感染者血样进行该方法的特异性验证;收集2011年1月至2012年9月浙江省各地妇幼保健医疗机构上送的96份婴幼儿样本,用新方法进行HIV的早期诊断,并将检测结果与罗氏HIV DNA定性检测试剂盒作比较。结果双重荧光PCR新方法能特异性检测HIV前病毒DNA,特异性为100％,检测灵敏度为100拷贝/反应;新方法和罗氏HIV DNA定性检测试剂盒检测96份婴幼儿样本,结果完全一致（符合率为100％）。结论建立的双重荧光PCR方法经济便捷、特异性好、灵敏度高、结果准确、易于推广,有望用于婴幼儿HIV早期诊断,并为HIV前病毒DNA的检测提供了一个通用技术平台。     

Development of real-time PCR assays for genotyping of Chlamydia trachomatis

We have developed and validated a nested real-time PCR (NRT-PCR) for the genotyping of Chlamydia trachomatis and used it specifically for the typing of either eight genovars from D to K or three genovars of lymphogranuloma venereum (LGV). The 11 probes used in the NRT-PCR correctly identified the DNA from D to K and LGV reference strains and did not cross-react with the DNA from 26 strains representing the bacterial pathogens and commensals of the oropharynx, genital tract, and rectum. The NRT-PCR had a 95% probability of detection at four genome copies (confidence interval, three to six copies) of C. trachomatis per reaction. One hundred cervical and urethral swab specimens containing C. trachomatis DNA from 63 women and 37 men were used to validate the method. The results from the NRT-PCR and the DNA sequencing of amplicons generated from the omp1 gene showed 100% correlation for these samples. The assay also identified the LGV-II genotype in 24 of 48 rectal swab specimens containing C. trachomatis DNA that were obtained from men having sex with men. The Sexually Transmitted Bacteria Reference Laboratory, London, independently confirmed these results using group-specific LGV real-time PCR and restriction fragment length polymorphism analysis. Compared with the NRT-PCR, non-NRT-PCR was found to be less sensitive: it typed C. trachomatis DNA in only 80% of the genital samples and 90% of the rectal swab samples. This is the first successful demonstration of the use of real-time PCR for the genotype-specific typing of C. trachomatis strains that cause sexually transmitted diseases.     

Use of polymerase chain reaction for detection of Chlamydia trachomatis.

A polymerase chain reaction (PCR) assay was developed for detection of Chlamydia trachomatis DNA. From the published sequence of the common C. trachomatis plasmid, two primer sets were selected. Detection of amplified sequences was done by agarose gel electrophoresis of cleaved or uncleaved amplified sequences, Southern hybridization, or dot blot analysis. The PCR assay was optimized and, after 40 cycles of amplification with primer set II, demonstrated a sensitivity of 10(-17) g of DNA, which corresponds to the detection of one copy of the plasmid. Because of the high sensitivity, we developed a closed system in which airborne contamination was minimized. Analysis of 228 clinical samples tested by cell culture, IDEIA enzyme immunosorbent assay (Medico-Nobel, Boots-Celltech Ltd., Berkshire, United Kingdom), and PCR showed a sensitivity of 100%, a specificity of 93% when PCR was compared with cell culture, and a corrected specificity of 99% when PCR was compared with cell culture or IDEIA.     

Characteristics of the m2000 automated sample preparation and multiplex real-time PCR system for detection of Chlamydia trachomatis and Neisseria gonorrhoeae

We evaluated a new real-time PCR-based prototype assay for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae developed by Abbott Molecular Inc. This assay is designed to be performed on an Abbott m2000 real-time instrument system, which consists of an m2000sp instrument for sample preparation and an m2000rt instrument for real-time PCR amplification and detection. The limit of detection of this prototype assay was determined to be 20 copies of target DNA for both C. trachomatis and N. gonorrhoeae, using serially diluted linearized plasmids. No cross-reactivity could be detected when 55 nongonococcal Neisseria isolates and 3 non-C. trachomatis Chlamydia isolates were tested at 1 million genome equivalents per reaction. Concordance with the Roche Amplicor, BDProbeTec ET, and Gen-Probe APTIMA Combo 2 tests was assessed using unlinked/deidentified surplus clinical specimens previously analyzed with these tests. For C. trachomatis, concordance for positive results ranged from 93.7% to 100%, while concordance for negative results ranged from 98.2% to 100%. For N. gonorrhoeae, concordance for positive and negative results ranged from 91.4% to 100% and 99.3% to 100%, respectively. A workflow analysis of the prototype assay was conducted to obtain information on throughput under laboratory conditions. At 48 samples/run, the time to first result for both C. trachomatis and N. gonorrhoeae was 4.5 h. A total of 135 patient specimens could be analyzed in 8.9 h, with 75 min of hands-on time. This study demonstrated the technical and clinical feasibility of the new Abbott real-time PCR C. trachomatis/N. gonorrhoeae assay.     

Detection of Chlamydia pneumoniae and Chlamydia psittaci in sputum samples by PCR.

AIMS--To use the polymerase chain reaction (PCR) to detect Chlamydia pneumoniae and Chlamydia psittaci in sputum samples. METHODS--A nested PCR was developed, the first stage of which amplified DNA from both C pneumoniae and C psittaci while the second stage targeted specifically at C pneumoniae, allowing the two species to be differentiated. The primers were designed not to amplify sequences from C trachomatis. A panel of 26 sputum samples from patients with community acquired pneumonia evaluated previously by enzyme linked immunosorbent assay (ELISA), direct immunofluorescence (DIF), and culture was tested blind by PCR. Most of these specimens also had accompanying serial serum samples which were tested for species specific antibodies using microimmunofluorescence (micro-IF). RESULTS--PCR detected C pneumoniae DNA in 10 of the 26 samples and C psittaci DNA in four. There was good concordance between ELISA, DIF, micro-IF and PCR in the C pneumoniae group. Two of the C psittaci identified by PCR were labelled C pneumoniae by DIF but the PCR results were supported by serology or a history of bird contact. Of the PCR negative group: six were true negative results; two contained C trachomatis. There were four discrepant results. CONCLUSIONS--The data suggest that PCR is effective in the detection of C pneumoniae. The sensitivity for C psittaci is inevitably lower due to the strategy taken but specificity seemed to be good.     

Touchdown enzyme time release-PCR for detection and identification of Chlamydia trachomatis, C. pneumoniae, and C. psittaci using the 16S and 16S-23S spacer rRNA genes

Three touchdown enzyme time release (TETR)-PCR assays were used to amplify different DNA sequences in the variable regions of the 16S and 16S-23S spacer rRNA genes specific for Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci as improved tests for sensitive diagnosis and rapid species differentiation. The TETR-PCR protocol used 60 cycles of amplification, which provided improved analytical sensitivity (0.004 to 0.063 inclusion-forming unit of Chlamydia species per PCR). The sensitivity of TETR-PCR with primer set CTR 70-CTR 71 was 96.7%, and the specificity was 99.6%, compared to those of the AMPLICOR PCR for the detection of C. trachomatis in vaginal swab samples. TETR-PCR for C. pneumoniae with primer set CPN 90-CPN 91 was 90% sensitive and 93.3% specific compared with a nested PCR with primer set CP1/2-CPC/D for clinical respiratory samples. TETR-PCR for C. psittaci with primer set CPS 100-CPS 101 showed substantial agreement with cell culturing (kappa, 0.78) for animal tissue samples. Primer sets were then combined into a single multiplex TETR-PCR test. The respective 315-, 195-, and 111-bp DNA target products were precisely amplified when DNA from each of the respective Chlamydia species or combinations of them was used. Multiplex chlamydia TETR-PCR correctly identified one strain of each of the 15 serovars of C. trachomatis, 22 isolates of C. pneumoniae, and 20 isolates of C. psittaci. The primer sets were specific for each species. No target products were amplified when DNA from C. pecorum or a variety of other microorganisms was tested for specificity. TETR-PCR with primers selected for specific sequences in the 16S and 16S-23S spacer rRNA genes is a valuable test that could be used either with individual primers or in a multiplex assay for the identification and differentiation of Chlamydia species from culture isolates or for the detection of chlamydiae in clinical samples.     

Detection of Chlamydia trachomatis by the polymerase chain reaction in swabs and urine from men with non-gonococcal urethritis.

A polymerase chain reaction (PCR) was developed for Chlamydia trachomatis in which a 380 base pair DNA fragment was amplified. Amplification occurred with the DNA from the 15 serovars but not with that from other Chlamydia spp or with DNA from a variety of other organisms. Chlamydial DNA (10(-16) g) could be detected and the PCR seemed to be able to detect single organisms. Urethral swabs were obtained from 37 men with acute non-gonococcal urethritis (NGU), 18 (49%) of whom were positive for C trachomatis by MicroTrak. As a result of clinical re-examinations 65 urethral swabs were available for analysis by the PCR. In comparison with MicroTrak, PCR had a sensitivity of 95%, a specificity of 94%, a positive predictive value of 86% and a negative predictive value of 98%. The PCR was apparently less sensitive (82%) in tests on urine samples. Overall, however, values of sensitivity and specificity of the PCR compared favourably with those of MicroTrak. The PCR for C trachomatis is likely to be a valuable technique for research, but problems of DNA contamination suggest that it should not be recommended for routine diagnosis.     

Application of a nested, multiplex PCR to psittacosis outbreaks.

We developed a nested, multiplex PCR for simultaneous detection of three species of chlamydiae in human and avian specimens. The PCR was designed to increase sensitivity and to circumvent inhibitors of PCR present in clinical specimens. The target sequence was the 16S rRNA gene. The first-step PCR was genus specific, and the second-step PCR was multiplexed (i.e., had multiple primer sets in the same tube) and could discriminate among Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia trachomatis on the basis of the molecular weight of the amplicon. The limit of detection of each of the two PCR steps was 5 inclusion-forming units. We used PCR and serologic evidence during outbreaks of psittacosis to infer that C. psittaci had been transmitted from birds purchased in pet stores to humans. We also used this method to test both live and dead birds from pet stores for infection with C. psittaci. Compared with culture, the application of PCR to avian specimens increased the rate of C. psittaci detection.     

mtLSU-巢式PCR检测实验大鼠卡氏肺孢子虫的实验研究

目的对mtLSU-巢式PCR方法检测大鼠卡氏肺孢子虫的应用价值以及基因序列进行评价。方法采用地塞米松免疫抑制法诱导大鼠感染肺孢子虫;实验组10只,对照组1只;诱导至第7周时收集实验组及对照组大鼠肺组织和支气管肺泡灌洗液（BALF）标本,采用mtLSU-巢式PCR方法对人源与鼠源肺孢子虫共有的基因进行扩增和序列测定,同时采用镜检法对实验组大鼠肺组织和肺泡灌洗液标本进行检测,评估两种方法的敏感性。结果采用mtLSU-巢式PCR方法对实验感染大鼠肺组织和BAL进行检测,卡氏肺孢子虫DNA阳性率分别为100%（10/10）、90%（9/10）。而GMS染色镜检法检测的阳性率分别为80%（8/10）、60%（6/10）。所测Wistar大鼠卡氏肺孢子虫mtLSU基因序列长度为155bp,与GenBank的大鼠源肺孢子虫（U20170）及人源肺孢子虫（DQ473446）同源性均为100%（154/154、155/155）。结论 mtLSU-巢式PCR方法应用于大鼠卡氏肺孢子虫检测敏感性高,特异性强;获得与人源耶氏肺孢子虫相同的Wistar大鼠卡氏肺孢子虫mtLSU的基因序列。     

Development and validation of a rotor-gene real-time PCR assay for detection, identification, and quantification of Chlamydia trachomatis in a single reaction

A multitarget real-time PCR (MRT-PCR) for detection of Chlamydia trachomatis DNA was developed and validated. There were three targets for amplification in a single reaction: the cryptic plasmid (CP), the major outer membrane protein (MOMP) gene, and an internal control. The assay had the following characteristics: (i) detection and confirmation of the presence of C. trachomatis DNA in a single reaction, (ii) detection of all genovars of C. trachomatis without any cross-reactivity with pathogenic bacteria or commensal organisms of the oropharynx and genital tract, (iii) a 95% probability of detection with three copies of MOMP and one copy of CP per reaction mixture, (iv) identification of the inhibition of amplification, (v) a quantitative dynamic range of 25 to 250,000 genome copies per reaction mixture, (vi) high intra- and interassay reproducibilities, and (vii) correct identification of all samples in the validation panel. There were 146 COBAS Amplicor PCR (Amplicor PCR)-positive samples and 122 Amplicor PCR-negative samples in the panel. MRT-PCR detected CP DNA alone in 6 (4%) Amplicor PCR-positive samples and both CP and MOMP DNAs in 140 (96%) of 146 Amplicor PCR-positive samples. The quantity of MOMP DNA in 95 (68%) of 140 samples was within the dynamic range of the assay. The median C. trachomatis load in these samples was 321 genome copies per reaction mixture (range, 26 to 40,137 genome copies per reaction mixture). Due to the inclusion of two different C. trachomatis-specific targets, the assay confirmed 259 (97%) of 268 results in a single reaction. This assay could be used in the qualitative format for the routine detection of C. trachomatis and in the quantitative format for study of the pathogenesis of C. trachomatis-associated diseases. The assay demonstrated the potential to eliminate the need for confirmatory testing in almost all samples, thus reducing the turnaround time and the workload.     

DNA sequencing validation of Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acid tests

DNA sequencing was used to confirm Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acids in endocervical swab samples. DNA in residues of the samples with positive results by 2 commercial kits was subjected to nested polymerase chain reaction (PCR) amplification. The nested PCR amplicons were used as templates for direct automated DNA sequencing. A 40-base signature sequence was sufficient to achieve unequivocal validation of C trachomatis cryptic plasmid and gonococcal opa gene DNA. DNA with a signature sequence specific for C trachomatis was identified in all 14 samples and for N gonorrhoeae in all 13 samples with positive results by the commercial kits for these 2 microbes. In a low-prevalence population, PCR retesting of 289 samples with initial negative results by a non-nucleic acid amplification assay revealed 3 samples positive for C trachomatis and 2 samples positive for N gonorrhoeae that were missed by the commercial kit. DNA sequencing is a useful tool in validating molecular diagnostics.