6.1 Best practices for collection and processing clinical specimens and extraction of RNA

Mick Mulders

The timely and proper collection and processing of virologic specimens from suspected cases of measles and rubella is crucial for the genetic characterization of measles and rubella viruses, and increasingly, to aid in case confirmation. Because the integrity of the viral RNA can be compromised at any point during specimen collection, processing, and transport, the procedures and recommendations described in Chapter 3. Clinical specimens for the laboratory diagnosis and molecular epidemiology of measles, rubella, and CRS should be followed to minimize degradation of the samples.

Oral fluid (OF), throat swabs (TS), or nasopharyngeal (NP) aspirates or swabs are good sources of RNA for measles or rubella virus detection [3,4,6]. Urine samples are acceptable but may be more difficult to transport and may contain substances that are inhibitory to RT-PCR. When a suspected measles or rubella case has been detected in an elimination setting, particularly cases that have no known source or contact with other cases, the collection of more than one clinical specimen may improve the likelihood of successful RNA detection and identification of the genotype.

Serum specimens (including eluate from dried blood spots) have been utilized as a source of virus-specific RNA when routine virologic specimens could not be obtained. However, successful detection of viral RNA in serum specimens requires that the blood sample is collected very close to rash onset (less than 3 days). Even when collected early, the number of virus particles in serum is low, making RNA detection very challenging. A protocol utilizing nested amplification primers can increase the sensitivity, but its use is limited as a last option for determination of the genotype (not for case confirmation) from specimens that had tested positive by real-time RT-PCR. This approach should only be used by laboratories with comprehensive containment and control procedures in place to mitigate any inadvertent cross-contamination.

The most important factor for successful amplification and identification of measles or rubella RNA by any detection method is the timing of the sample collection. The specimens should be collected as soon as possible after rash onset as the sensitivity of detection decreases with time after rash onset. The recommended timing for collection of clinical specimens is provided in Chapter 3. The sensitivity of RNA detection methods from most types of specimens declines considerably after 5-7 days. As discussed in Chapter 3. Clinical specimens for the laboratory diagnosis and molecular epidemiology of measles, rubella, and CRS, different types of clinical specimens may be collected beyond 5 days post rash onset for detection of measles RNA if a delay is unavoidable. Successful amplification of measles virus-specific RNA by real-time RT-PCR from oral fluid specimens collected as late as 21 days after rash onset has been reported [2].

Upon arrival at the laboratory, the contents of the package containing the clinical specimen for RT-PCR testing is inspected and the condition of the clinical specimen is recorded (see Chapter 11. Data management and reporting of laboratory results). If not accessioned and processed immediately, the specimens should be transferred to either a refrigerator (4°-8°C) or frozen at -70°C, according to the SOP for the type of specimen. However, repeated cycles of freezing and thawing should be avoided to preserve the integrity of the RNA.

The extraction of RNA from a clinical specimen (or cell culture lysate) must be performed carefully to maximize the yield of target RNA. The best approach for RNA extraction is to use one of the commercial RNA extraction kits (e.g. Qiagen) or commercially prepared reagents. RNA extraction protocols for clinical specimens that are in use by GMRLN laboratories are available in Annex 6.1. When choosing an RNA extraction method, it is important to consider the source and type of specimen. For example, RNA extraction kits that are designed to extract RNA directly from clinical specimens may be unsuitable for extracting RNA from virus-infected cell culture lysates.