Multiplex detection for gastroenteritis pathogens

The gastroenteritis-causing microbes are spread via the faecal–oral route and infect from human-to-human or via food or drinking water contaminated with sewage water or human handling. Many pathogens cause gastroenteritis (see Table 1).

Nucleic acid-detecting technologies are, alone or as complement to culture on selective media, microscopy, or antigen detection, becoming standard in clinical microbiology laboratories. The techniques for detecting, identifying and quantifying the microbes concomitantly with their antimicrobial resistance or toxin genes are improving, but are still mostly limited to methods for detection of single, or a few, pathogen species. The target variation and horizontal gene transfer are confounding factors. Multiplex detection refers to a simultaneous detection and identification of many targets in the same or parallel analysis of one sample.

The requirements of diagnostic tests are high sensitivity, specificity and reproducibility. The assay should also be quick, inexpensive and require few hands-on steps. A sample can be divided and run in consecutive single tests to rule out different pathogens, or into parallel single tests using, for example, the same polymerase chain reaction (PCR) programme profile. The benefits of multiplex tests compared with a single test are: 

• Quicker results
• More cost effective 
• More efficient use of the sample
• Detection of co-infections
• Reduction of the unnecessary use of antibiotics
• Detection of non-prioritised microbes, which gives a better understanding of their epidemiology
• Simultaneous detection of virus, bacteria, parasites and fungi, covering a larger part of clinical microbiology. 

Commercial multiplex tests are often expensive per sample, but cost effective per pathogen. An ‘in house’ multiplex test is expensive to develop because it takes both skill and time. When up and running, the ‘in-house’ test is cost effective and it has the advantage of being flexible and easy to update. The multiplex design procedure consists of a loop of redesign for each addition a new primer-probe set:  

• Identifying a target-specific region
• Adjusting primer and probes for compatible Tm and Delta G
• Avoiding primer and probe monomers, hairpin-structures, heterodimers (especially amplifiable)
• Avoiding cross-hybridisation to the human genome, for example.

The risk of formation of primer–dimers increases as the panel expands and reduces the sensitivity of the assay. Utilising databases and programs such as BlastN (NCBI, NIH),(1) ClustalX,(2)  ConSort©, Mfold,(3) VisualOmp (DNASoftware)(4) and NucZip(5) can be useful tools. 

Viruses, especially RNA viruses, are difficult to detect because of a high genetic variability and diversity. This is caused by a ‘sloppy’ polymerase with a high mutational rate.

A thorough bioinformatic analysis of the distribution of variation across the target sequence is necessary. To gain specificity and sensitivity, the viral primers often have to contain degenerations. Having several wobbles creates a cocktail of primers with a decreased concentration of each specific primer, but the chance of having a more or less perfect match increases. The recently published NucZip algorithm(5)  describes how to achieve variation tolerance based on enumeration of long, perfectly matching segments by mapping the frequency and pattern of the nucleotide variation in a particular pathogen and by incorporating LNA, deoxy Inosines (dInosines) and degenerated nucleotides in suitable positions. 

Bacteria and fungi have very conserved genomes. To find a target-specific sequence that distinguishes closely related species from each other, and that separates a pathogenic species from one that is part of the normal flora, can be difficult. This feature can be used in a multiplex design strategy by placing primers in conserved regions and a probe in a variable region between the primers, reducing the amount of different primers and minimising primer–dimer formation.

All multiplex nucleic acid methods are based on nucleic acid extraction and amplification, followed by detection and identification. Nucleic acid extracted from a faecal sample contains a mix of RNA and DNA from the normal flora of bacteria, human cells and the pathogens, together with inhibitors affecting the efficiency of amplification.

The strategy of amplification and detection can be made with many different primer and probe techniques. The primers can be target-specific or consist of a random oligonucleotide sequence, which could then be combined with a generic sequence into one oligonucleotide, act separately in a consecutive order, or be linked as a padlock-probe. The primers and probes can be degenerated or contain nucleotide analogues, such as dInosines, that hybridise to all four types of nucleotide bases. 

Different techniques can be used to detect and identify the amplicon, such as size separating gel electrophoresis, real-time PCR, melting point analysis, microarray (solid phase or bead liquid array) or sequencing. If an intercalating dye such as ethidium bromide or an isotope is used, the amplicons are detected and identified by size with gel electrophoresis. The size differences have to be distinguished easily on the gel, which often limits the method to six-to-ten targets in one panel.(6,7)

Intercalating dyes, such as SYBRGreen or Eva Green, used in a real-time instrument will not identify what is amplified (target or primer–dimer), and are not suitable for multiplexing unless a melting curve analysis is applied after the amplification. A Taqman probe has a fluorophore in its 5’ end, the signal of which is quenched by an internal or 3’ end quencher. The polymerase (having a 5’ to 3’ exonuclease activity) cleaves off the 5’ fluorophore and the PCR instrument detects it. New probe binds in for each cycle, is cleaved, and the signal increases exponentially with the amount of specific amplicons produced.

Different fluorophores, combined with specific quenchers, can be used in different probes to create a multiplex real-time PCR,(8) but the multiplexity is limited to approximately four-plex by the PCR instrument’s ability to distinguish the somewhat overlapping fluorescent signals. An example is the FTD Gastroenteritis kit (Fast-track Diagnostics, Luxembourg). It detects five viruses in two panels of Taqman RT-PCR, whereas Diagenode (Liège, Belgium) divides their multiplex Taqman PCR into four reactions with two-to-three pathogens.

High multiplexity can be gained by hybridising the amplicons to a microarray with millions of probes,(9–11) but the method has limited sensitivity and is time consuming because of slow diffusion times. The PCR product can be labelled with biotinylated nucleotides or primers, and detected with the streptavidin-linked reporter dye R-phycoerythrin after hybridisation to a probe linked to a solid phase microarray or to a bead in liquid bead arrays, as in the Luminex system(12–14) (Luminex Corporation, Austin, Tx). Suspension arrays have a shorter hybridisation time because bead-bound probes have a higher likelihood of a collision with a target. 

Multiplex gastroenteritis methods have been developed using different strategies of amplification and read-out platforms.(6–14) Seegene (Seoul, Korea) is amplifying with DPO™ (Dual Priming Oligonucleotide) technology and detects with a size-separating capillary electrophoresis. Seeplex® Diarrhea ACE Detection detects 15 pathogens, divided into three panels. The DPO™ technology divides the primer in two sections with the help of dInosines, creating a 3’ short target specific end and a 5’ long anchoring target specific sequence. The analysis of the endpoint PCR on gel is a risk for contamination. 

Recently Ohrmalm et al published a new method called variation tolerant capture multiplex assay, VOCMA,(15) which was applied for a 7-plex virus gastroenteritis VOCMA. The variation tolerant primers, called primer-probes, were designed according the NucZip-algorithm.(5) They comprise a short generic sequence at the 5’ end and 50–70 nucleotide-long target specific sequence at the 3’ end (Figure 1). The assay is a one-step, one-tube multiplex PCR with a low concentration of the primer-probes, which minimises the amount of primer–dimer formation, together with a higher concentration of generic primer; amplification controlled with a temperature switch. The biotinylated amplimers are detected and identified in the Luminex system with xMAP bead linked variation tolerant detection probes targeting the region between the primer-probe target sequences. 

The xTAG® Gastrointestinal Pathogen Panel (xTAG GPP) from Luminex detects 15 major gastrointestinal pathogens with the xTAG technology. One of the primers has a unique tag, which allows the biotinylated product to be hybridised to an anti-tag coupled to the color-coded bead. The transferring of post-PCR amplicons, in both the xTAG GPP and VOCMA, to the hybridisation mixture is a contamination risk and none of the methods is quantitative.

PathoFinder (Maastricht, Netherlands) and Seegene (Korea) use melting curve analysis in a real-time PCR instrument with different fluorophores and probe techniques for their 22 and 16 pathogen-detecting respiratory panels, respectively, and avoid the post-PCR opening of the tube. These technologies have not yet been applied for gastroenteritis. 

The number of publications about gastroenteritis using the multiplex detection assays, having different benefits and drawbacks, are increasing. The combination of a quick, highly multiplex, yet highly sensitive, method with a closed post-PCR system has still to come. 

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