比较疟疾病例样本套式PCR检测和传统镜检结果，探讨套式PCR在疟疾诊断中的应用价值。

采集疟疾患者血样，分别涂制厚、薄血膜用于常规镜检；抗凝血或滤纸血用于提取疟原虫DNA等。按要求设计属种等多套引物，应用套式PCR扩增感染人的4种疟原虫目的基因片段，将检测结果与镜检结果进行比较分析，并对目的片段进行测序鉴定等。

259份血样，套式PCR共检测出恶性疟187例、间日疟45例、卵形疟8例和三日疟1例，阴性10例。其中恶性疟阳性检出率最高(75.10%)，间日疟阳性检出率次之(18.07%)，还检测到混合感染血样8份(3.21%)。与GenBank标准序列对比显示，扩增片段大小及测序结果均完全正确，显示套式PCR在分型上比传统镜检更客观。套式PCR检测与传统镜检结果比较显示，前者阳性率(96.14%)明显高于后者(90.73%)，并且差异具有统计学意义(χ²＝12.07，P<0.05)。

套式PCR法检测疟原虫具有较高敏感性和特异性，且适用于疟原虫混合感染，在疟疾病例诊断和分型方面均具有良好的应用前景。

To compare the performance of nested PCR assay with traditional microscopic examination of Giemsa-stained blood films and to discuss the value of nested PCR application for the diagnosis of malaria.

Blood samples were collected from affiliated hospital of Shandong Institute of Parasitic Diseases and 17 cities′ CDC reported cases in Shandong province. Finger prick blood samples were taken from each participating individual and used to prepare Giemsa-stained thick and thin blood films for traditional microscopy. Anticoagulant or filter paper blood samples were used to extract DNA from malaria parasites.Primers of plasmodium genus-species were designed on the basis of conserved sequence on 18S small subunit ribosomal RNA(SSU rRNA). Nested PCR was applied to the amplification of target gene segments on 4 kinds of plasmodium infected human. The results of PCR were compared with microscopy and were processed by statistical analysis (chi-square test). The target fragments of PCR amplification were used for species identification and compared with the standard sequence registered in Genbank.

Among the 259 samples, 249 had been detected positive for malaria parasites by nested PCR. The electrophoresis banding size of 187 samples were 205bp, identified as plasmodium falciparum. The electrophoresis banding size of 45 samples were 120bp, identified as plasmodium vivax.The electrophoresis banding size of 8 samples were 800bp, identified as plasmodium ovale. The electrophoresis banding size of 1 sample was 144bp, identified as plasmodium malariae. No species amplification bands were found in 10 samples, identified as negative. Plasmodium falciparum occupied the highest detection rate of positive samples (75.10%) and plasmodium vivax occupied the second highest detection rate of positive samples (18.07%), in addition, 8 mixed infection were detected (3.21%). PCR products of 5 plasmodium falciparum, 5 plasmodium vivax, 4 plasmodium ovale and 1 plasmodium malariae were sequencing and compared with standard sequence registered in Genbank by blast.The comparison results showed that fragments size and product sequence was in accordance with the reference. Microscopy results showed that 171 samples were plasmodium falciparum, 60 samples were plasmodium vivax, 4 samples were plasmodium ovale, 24 samples were negative. Species proportion of microscopy results were similar to nested PCR, plasmodium falciparum occupied the highest detection rate of positive samples (72.77%), plasmodium vivax occupied the second highest detection rate of positive samples (25.53%). However, there was no plasmodium malariae and mixed infection samples detected by microscopy. Nested PCR were compared with traditional microscopy and the results showed that consistent rate of detected samples between 2 methods was 84.56%.But nested PCR positive rate(96.14%) was significantly higher than traditional microscopy(90.73%), the difference between 2 groups had statistical significance (χ²=12.07, P<0.05). Fourteen samples were detected positive by nested PCR but negative by microscopic examination.Ten samples were detected negative by the both methods.It was worth noting that no samples detected positive by microscopic examination but negative by nested PCR.Furthermore, all 4 kinds of plasmodium species and mixed infection were detected by nested PCR but only 3 kinds of plasmodium species without mixed infection were detected by traditional microscopic examination.The results showed that nested PCR was more objective than the traditional microscopy in malaria parasites′ classification.

The results indicate that nested PCR used for detection of malaria parasites with high sensitivity and specificity and capable of identifying malaria parasites at the species level when microscopy is equivocal. Nested PCR is also suitable for the mixed infection of malaria parasites and has a broad application prospect in the study of diagnosis and molecular epidemiology of malaria cases.