应用液相芯片技术检测拉米夫定耐药相关性HBV变异

Detection of lamivudine resistance-associated hepatitis B virus mutations by multi-analyte suspension array

目的建立一种检测拉米夫定（LAM）耐药相关性HBV变异的检测系统。方法从血清样本中提取病毒DNA，经纯化后，与生物素标记的引物经过PCR扩增后，来自HBV开放阅读框架的、被生物素标记的DNA扩增产物与偶联在微球上的特异性寡核苷酸探针杂交，未能结合微球的DNA被洗脱，加入标记有链亲素修饰的藻红蛋白（SAPE），在Luminex分析仪上，用双色激光分别激发微球的红色荧光（定性）和SAPE的绿色荧光（定量），从而判断靶基因的突变类型和相对含量。采用液相芯片（muhi-analyte suspension array，MASA）技术构建有LAM相关耐药的HBV全基因质粒和25例LAM耐药的慢性乙型肝炎患者（CHB）的HBV逆转录区（RT）第180和204位氨基酸的变异情况；并与普通测序法相比较，以了解该方法的特异性；同时将变异株和野毒株按不同比例混合后检测，以了解该方法的敏感性。结果MASA可同时检测a180和n204氨基酸变异，并能准确判断变异的类型及混合变异；MASA对25例LAM耐药的CHB患者相关耐药，检测发现有15份患者标本中20411探针杂交信号明显升高（测序变异阴性和阳性荧光值中位数分别为5和106），有11份样本与204V探针杂交信号较高（测序变异阴性和阳性荧光值中位数分别为9和114）差异有统计学意义（z=-4．588，P〈0．05；z=-4．520，P〈0．05）；有2份发现2种探针杂交信号均很高，即有YIDD和YVDD混合变异株存在；仅有1份标本与20412探针杂交信号明显较高。同样用该方法检测20份未治疗患者的血清标本，均未发现LAM相关性变异。当病毒群中含有大于5％的LAM耐药相关HBV变异株时，即可被本方法检测到。结论MASA可用于LAM相关耐药变异株的早期检测。该方法较普通测序法灵敏，且操作简便、高通量、需要时间短，便于临床大样本检测的应用。

Objective To develop and establish a rapid, convenient method with high-throughput, high sensitivity and specificity for detection of lamivudine （ LAM ） resistance-associated mutations in the hepatitis B virus. Methods HBV DNA was extracted as PCR templates. The target sequence at RT region was amplified using primers with 5 ＇-biotin label. The biotin-labelled target PCR products interacted with those bead-bound probes in aqueous suspension, and then streptomycin and avidin-modified straptavidin phycoerythrin （SAPE） were added to the aqueous suspension after the free HBV DNA was washed out, which had not hybridized with probes on the beads. Finally, mutations were tested in luminex analyzer using the bicolor laser （red and green laser）. The mutated type and relative quantity were identified according to the different ratios of red fluorophores incorporated into the beads （for qualitative analysis ） and green fluorophores imparted by a specific reporter molecule SAPE （ for quantitative analysis）. Subsequently, the multi-analyte suspension array （MASA） was applied to detect plasmids containing full-length HBV genome with LAM-resistance associated HBV mutations and 25 serum specimens from patients with chronic hepatitis B, who were treated with LAM and experienced virological breakthrough due to LAM-resistance. The specificity of this method was compared with direct DNA sequencing. Meanwhile, we also determined the sensitivity of this method by mixing the wild and mutant type plasmids in different proportions. Results MASA can rapidly detect mutations at rt180 and rt204 positions in RT region simultaneously. 25 samples with LAM- resistant mutations confirmed by DNA sequencing were found to contain consistent mutations by this method, with 15 cases hybridizing to a particular 20411 probe type（negative and positive median signal values were 5 and 106 respectively） , 11 cases hybridizing to 204V probe（ negative and positive median signal values were 9 and 114 respectively） （Z value = - 4. 588 and - 4. 520, P 〈 0. 05 ）, and only 1 specimen hybridizing to 20412 probe. However, two samples were found harboring mixed mutated types （rtM204V and rtM204I）. More importantly,MASA was able to detect 5% mutation frequency with up to 100% of specificity. Conclusions MASA could be used for early detection of LAM resistance-associated mutations in hepatitis B virus. This method has high sensitivity, specificity and high-throughput in detection of LAM resistance-associated mutations compared with direct DNA sequencing. This method may also significantly save the time of diagnosis and facilitate the clinical application of large samples.