Morré S A, Sillekens P, Jacobs M V, van Aarle P, de Blok S, van Gemen B, Walboomers J M, Meijer C J, van den Brule A J
Department of Pathology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands.
J Clin Microbiol. 1996 Dec;34(12):3108-14. doi: 10.1128/jcm.34.12.3108-3114.1996.
In the present study, the suitability of RNA amplification by nucleic acid sequence-based amplification (NASBA) for the detection of Chlamydia trachomatis infection was investigated. When comparing different primer sets for their sensitivities in NASBA, use of both the plasmid and omp1 targets resulted in a detection limit of 1 inclusion-forming unit (IFU), while the 16S rRNA appeared to be the most sensitive RNA target for amplification (10(-3) IFU). In contrast, for DNA amplification by PCR, the plasmid target was optimal (10(-2) IFU), which is 10 times less sensitive than rRNA NASBA. To exclude false negativity in NASBA detection because of inhibition of amplification and/or inefficient sample preparation, an internal standard was developed. The internal control was added prior to sample preparation. This 16S rRNA NASBA with an internal control was compared with a plasmid DNA PCR by using a group of C. trachomatis-negative (n = 41) and -positive (n = 37) cervical scrapings, as determined by enzyme immunoassay (EIA). In addition, urine samples from the EIA-positive women were tested (n = 17). Both NASBA and PCR assays were able to detect C. trachomatis in all EIA-positive cervical scrapings, the corresponding urine samples, and two samples from the EIA-negative group. The internal NASBA standard was found clearly in all EIA-negative samples. In conclusion, these results indicate that detection of C. trachomatis by RNA amplification by NASBA with an internal standard is a suitable and highly sensitive detection method, with potential use in the diagnosis of urogenital C. trachomatis infections with cervical scrapings as well as urine specimens.
在本研究中，调查了基于核酸序列扩增（NASBA）的RNA扩增用于检测沙眼衣原体感染的适用性。在比较不同引物组在NASBA中的敏感性时，使用质粒和omp1靶标均能实现1个包涵体形成单位（IFU）的检测限，而16S rRNA似乎是最敏感的RNA扩增靶标（10⁻³ IFU）。相比之下，对于通过PCR进行的DNA扩增，质粒靶标是最佳的（10⁻² IFU），其敏感性比rRNA NASBA低10倍。为排除NASBA检测中由于扩增抑制和/或样本制备效率低下导致的假阴性，开发了一种内标。在样本制备前加入内对照。通过使用一组经酶免疫测定（EIA）确定为沙眼衣原体阴性（n = 41）和阳性（n = 37）的宫颈刮片，将这种带有内对照的16S rRNA NASBA与质粒DNA PCR进行比较。此外，对EIA阳性女性的尿液样本（n = 17）进行了检测。NASBA和PCR检测均能在所有EIA阳性的宫颈刮片、相应的尿液样本以及EIA阴性组的两个样本中检测到沙眼衣原体。在所有EIA阴性样本中均清晰地检测到了NASBA内标。总之，这些结果表明，采用带有内标的NASBA进行RNA扩增检测沙眼衣原体是一种合适且高度灵敏的检测方法，在诊断宫颈刮片及尿液标本的泌尿生殖道沙眼衣原体感染方面具有潜在应用价值。
