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Standardisation and evaluation of a quantitative multiplex real-time PCR assay for the rapid identification of Streptococcus pneumoniae

机译：用于快速鉴定肺炎链球菌的定量多重实时PCR分析的标准化和评估

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Rapid diagnosis of Streptococcus pneumoniae can play a significant role in decreasing morbidity and mortality of infection. The accurate diagnosis of pneumococcal disease is hampered by the difficulties in growing the isolates from clinical specimens and also by misidentification. Molecular methods have gained popularity as they offer improvement in the detection of causative pathogens with speed and ease. The present study aims at validating and standardising the use of 4 oligonucleotide primer-probe sets (pneumolysin [ ply ], autolysin [ lytA ], pneumococcal surface adhesion A [ psaA ] and Spn9802 [DNA fragment]) in a single-reaction mixture for the detection and discrimination of S. pneumoniae . Here, we validate a quantitative multiplex real-time PCR (qmPCR) assay with a panel consisting of 43 S. pneumoniae and 29 non-pneumococcal isolates, 20 culture positive, 26 culture negative and 30 spiked serum samples. A standard curve was obtained using S. pneumoniae ATCC 49619 strain and glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) gene was used as an endogenous internal control. The experiment showed high sensitivity with lower limit of detection equivalent to 4 genome copies/μl. The efficiency of the reaction was 100% for ply , lytA , Spn9802 and 97% for psaA . The test showed sensitivity and specificity of 100% with culture isolates and serum specimens. This study demonstrates that qmPCR analysis of sera using 4 oligonucleotide primers appears to be an appropriate method for the genotypic identification of S. pneumoniae infection.
机译：肺炎链球菌的快速诊断可以在降低感染的发病率和死亡率中发挥重要作用。肺炎球菌疾病的准确诊断受制于从临床标本中分离出的分离物的困难以及错误的识别。分子方法已迅速普及，因为它们提供了对致病性病原体检测的改进。本研究旨在验证和标准化在单反应混合物中4种寡核苷酸引物探针组（肺炎球菌溶血素[ply]，自溶素[lytA]，肺炎球菌表面粘附A [psaA]和Spn9802 [DNA片段]）的使用。肺炎链球菌的检测与鉴别。在这里，我们验证了由43个肺炎链球菌和29个非肺炎球菌分离株，20个培养阳性，26个培养阴性和30个加标血清样品组成的小组的定量多重实时PCR（qmPCR）分析。使用肺炎链球菌ATCC 49619菌株获得标准曲线，并将3-磷酸甘油醛脱氢酶（GAPDH）基因用作内源性内部对照。实验显示出高灵敏度，检测下限相当于4个基因组拷贝/μl。对于ply，lytA，Spn9802，反应效率为100％，对于psaA，反应效率为97％。该测试显示对培养分离物和血清标本的敏感性和特异性为100％。这项研究表明，使用4种寡核苷酸引物对血清进行qmPCR分析似乎是鉴定肺炎链球菌感染的基因型的合适方法。

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