Department of Tropical Medicine, Tulane University, New Orleans, LA 70112, USA.
Malar J. 2012 Jun 10;11:193. doi: 10.1186/1475-2875-11-193.
Reliable methods to preserve mosquito vectors for malaria studies are necessary for detecting Plasmodium parasites. In field settings, however, maintaining a cold chain of storage from the time of collection until laboratory processing, or accessing other reliable means of sample preservation is often logistically impractical or cost prohibitive. As the Plasmodium infection rate of Anopheles mosquitoes is a central component of the entomological inoculation rate and other indicators of transmission intensity, storage conditions that affect pathogen detection may bias malaria surveillance indicators. This study investigated the effect of storage time and temperature on the ability to detect Plasmodium parasites in desiccated Anopheles mosquitoes by real-time polymerase chain reaction (PCR).
Laboratory-infected Anopheles stephensi mosquitoes were chloroform-killed and stored over desiccant for 0, 1, 3, and 6 months while being held at four different temperatures: 28, 37, -20 and -80°C. The detection of Plasmodium DNA was evaluated by real-time PCR amplification of a 111 base pair region of block 4 of the merozoite surface protein.
Varying the storage time and temperature of desiccated mosquitoes did not impact the sensitivity of parasite detection. A two-way factorial analysis of variance suggested that storage time and temperature were not associated with a loss in the ability to detect parasites. Storage of samples at 28°C resulted in a significant increase in the ability to detect parasite DNA, though no other positive associations were observed between the experimental storage treatments and PCR amplification.
Cold chain maintenance of desiccated mosquito samples is not necessary for real-time PCR detection of parasite DNA. Though field-collected mosquitoes may be subjected to variable conditions prior to molecular processing, the storage of samples over an inexpensive and logistically accessible desiccant will likely ensure accurate assessment of malaria parasite presence without diminishing PCR-detection of parasites in mosquitoes stored for at least six months.
为了检测疟原虫，需要可靠的方法来保存蚊虫媒介用于疟疾研究。然而，在野外环境中，从采集到实验室处理的过程中保持冷链储存，或者获得其他可靠的样本保存方法，在后勤上往往不切实际或成本过高。由于按蚊感染疟原虫的比率是昆虫接种率和其他传播强度指标的核心组成部分，因此影响病原体检测的储存条件可能会使疟疾监测指标产生偏差。本研究通过实时聚合酶链反应（PCR）调查了储存时间和温度对干燥按蚊中检测疟原虫能力的影响。
用氯仿杀死感染的实验室按蚊斯蒂芬斯（Anopheles stephensi），并用干燥剂储存，在 28、37、-20 和-80°C 四个不同温度下储存 0、1、3 和 6 个月。通过实时 PCR 扩增裂殖体表面蛋白第 4 块的 111 个碱基对区域评估疟原虫 DNA 的检测。
干燥蚊虫的储存时间和温度变化并不影响寄生虫检测的敏感性。双因素方差分析表明，储存时间和温度与寄生虫检测能力的丧失无关。在 28°C 下储存样本可显著提高检测寄生虫 DNA 的能力，尽管在实验储存处理和 PCR 扩增之间没有观察到其他阳性关联。
对于实时 PCR 检测寄生虫 DNA，无需对干燥的蚊虫样本进行冷链维护。尽管在进行分子处理之前，野外采集的蚊虫可能会受到不同条件的影响，但在经济实惠且易于获取的干燥剂上储存样本，很可能会确保对疟原虫存在的准确评估，而不会降低对储存至少六个月的蚊虫中寄生虫的 PCR 检测能力。
