A 10 Year Analysis of the Use of Multiplex Real-Time PCR Screening for Botulinum Neurotoxin-Producing Clostridium Species

Research Article

J Bacteriol Mycol. 2016; 3(3): 1030.

A 10 Year Analysis of the Use of Multiplex Real-Time PCR Screening for Botulinum Neurotoxin-Producing Clostridium Species

Davis SW1, Kelly-Cirino CD2, Cirino NM3, Hannett GE4, Musser KA4 and Egan C1*

1Biodefense Laboratory, Wadsworth Center, New York State Department of Health, USA

2DNA Genotek, Canada

3Department of Medicine, University Hospitals, Case Western Reserve University, Cleveland, USA

4Bacteriology Laboratory, Wadsworth Center, New York State Department of Health, USA

*Corresponding author: Egan C, Biodefense Laboratory, Wadsworth Center, New York State Department of Health, USA

Received: July 11, 2016; Accepted: August 02, 2016; Published: August 04, 2016


Prompt testing of specimens suspected of containingbotulinum neurotoxins produced by Clostridium botulinum, C. butyricum, and C. baratiiisis essential due to the potential of these toxins to produce rapid morbidity and mortality in humans. The Standard Mouse Bioassay (SMB) is the gold standard for C. botulinum neurotoxin (BoNT) testing but has several limitations including a long, labor intensive testing process and difficult result interpretation. We have developed and evaluated a sensitive screening tool for the detection of Clostridium spp. neurotoxin genes, BoNT types A, B, E, F and C. baratii F, by real-time PCR using an automated DNA extraction. Clinical specimens and environmental samples were analyzed over a 10-year period by real-time PCR, SMB, and culture. A total of 61 cases, clinically compatible with foodborne or infant botulism were submitted to our laboratory between 2003-2013. PCR was positive for 31 cases, 81% of these were confirmed by culture and 45% were confirmed by SMB. We also found that screening specimens by PCR provides an early indication of botulism in approximately 4 hours on all specimen types. Comparatively, SMB testing requires at least four days, is not appropriate for all specimen types, and requires sufficient quantities of specimen for analysis which precluded its use in 28 cases in our study. This rapid, high-confidence, cost-effective assay that detects the major BoNT types is a great addition to the investigation of suspect cases of botulism.

Keywords: Clostridium botulinum; Botulinum toxin; Botulism; Real-time PCR; Multiplex PCR


ATCC: American Tissue Type Culture Collection; BoNT: C. botulinum Neurotoxin; CBI: C. botulinum Isolation Agar; CFU: Colony Forming Units; LOD: Limit Of Detection; NYSDOH: New York State Department of Health; spp: Species; PCR; Polymerase chain reaction; SMB: Standard Mouse Bioassay; TPGY: Trypticase- Peptone-Glucose-Yeast-Extract


Clostridium botulinum, C. baratii, and C. butyricum are gram positive, anaerobic bacteria which may produce potent neurotoxins. Rapid clinical testing is critical because intoxication from BoNT types A, B, E, F, and C. baratii F can result in fatal illness in humans [1]. The CDC case definitions of botulism as of 2011 for foodborne botulism and infant botulism have been described [2]. A confirmed case of foodborne botulism is a clinically compatible case that is laboratory confirmed or that occurs among a person who ate the same food as persons who have laboratory-confirmed botulism. The laboratory criteria for diagnosis include the detection of botulinum toxin in serum, stool or patient’s food or the isolation of C. botulinum from stool. There is a similar definition for infant botulism however the case must be in a child less than 1 year of age and detection in food is not relevant.

The standard mouse bioassay (SMB), which detects active toxin, is the “gold standard” for BoNT testing [3]. This method has several limitations including being very labor intensive, expensive, ethically challenging due to the use of live mice, and having the potential of producing false positive results [4]. Even though the use of the SMB has been much debated, particularly for comparison of toxin preparations in clinical use, this assay utilizing mouse lethality remains the standard for detection and quantitation of in-vivo neurotoxicity [5,6,7]. Other methods of identification of toxicity such as those determining immunological reactivity or catalytic activity need to be more extensively compared to the mouse bioassay before they are adopted for general use [8].

Polymerase Chain Reaction (PCR) assays have been developed and utilized in BoNT testing previously [9,10]. The few reports that have been published have evaluated foods and fecal material using PCR based methods but none used real-time testing directly on clinical specimens. Also, the published assays have not included all of the most relevant BoNT types detected in North America (including C. baratii toxin type F) that cause the majority of botulism cases in a multiplex format nor did they utilize laboratory automation that is beneficial for large outbreaks or response to potential bio-threat events.

Real-time PCR testing is also valuable where there is insufficient volume of clinical specimen for direct mouse bioassay testing. In these situations, culture and isolation of the toxigenic bacteria must be performed before any indication of botulism is detected or confirmed in the laboratory thus delaying the identification of botulism which can lead to a serious impact to patient treatment and care. Culture and isolation of bacterial colonies from primary specimens is time consuming and often results in having to test many suspect colonies that have similar morphology to C. botulinum. In addition, there may be Clostridium spp. present in stool that do not produce toxin. When testing for infant botulism, specimens are frequently received in the laboratory that does not meet the minimum volume criteria for SMB testing. PCR testing is extremely useful in these situations as very small volumes can be successfully testing using this method. Botulism can cause paralysis of the GI tract and thus infants cannot produce the necessary specimen quantity needed for the SMB. Often, diagnosis by molecular methods is the only mechanism by which a highly reliable and sensitive result is obtained within a time frame relevant for providing meaningful results to physicians and public health epidemiologists.

We developed a real-time PCR screening assay using automated nucleic acid extraction from specimens which specifically detects DNA from C. botulinum types A, B, E, F, and C. baratii F (BoNT real-time PCR). The real-time PCR can be utilized directly on stool, rectal swabs, and enema specimens and results can be obtained in <5 hrs. This new assay was evaluated with SMB and culture on 61 clinical specimens received at the Wadsworth Center, New York State Department of Health (NYSDOH) over a 10-year period. This study was designed to determine if this new multiplex real-time PCR assay is an acceptable screening tool that can be used by public health and/ or clinical laboratories for rapid detection of botulism cases.

Materials and Methods

Bacterial strains

All strains utilized in the testing including C. botulinum and C. baratii strains were obtained from the culture collection at the Wadsworth Center, New York State DOH or the American Tissue Type Culture Collection (ATCC). C. botulinum and C. baratii were grown at 37°C overnight on sheep blood agar plates or CDC anaerobe agar plates. An anaerobic environment was created using Gas Pack jars utilizing AnaeroGen (Oxiod) or an anaerobic chamber (Coy Labs).

C. botulinum and C. baratii culture from stool specimens

Stool samples were cultured by inoculation to C. botulinum Isolation agar (CBI) [11]. Plates were incubated for at least 48 h at 37°C. For isolation of C. botulinum, lipase positive colonies were picked and identified using conventional biochemical reactions. C. baratii was isolated by picking colonies that showed a characteristic narrow zone of lecithinase on CBI. Toxin production of isolates was tested by cultivation of the organism in Trypticase-Peptone-Glucose- Yeast-extract (TPGY) broth [12] for 48h. The broth was filtered by passing through a 0.45 micron filter. SMB was performed on filtrates in the same manner as stool samples. Selected stool specimens were enriched by inoculation of stool in TPGY. Broths were incubated in an anaerobic atmosphere for 48h at 37°C.

Extraction of clinical specimens

Clinical stool specimens were obtained from the NYSDOH Bacteriology Laboratory. Pretreatment to remove solid materials from stool specimens was required before performing the DNA extraction as per the manufacturer. Stool specimens and environmental samples were diluted in Tris EDTA buffer to obtain 200ul of liquid content, mixed thoroughly and heat treated at 95°C for 30min. The specimen was then centrifuged for 10s at 14,000 RPM and the supernatant was recovered. The supernatant was utilized for nucleic acid extraction by either manual or automated methods. Manual extraction was performed using the Master Pure complete DNA and RNA Purification Kit (Epicentre Technologies) with the modification of a 30min lysis step followed by filtering in a spin module column (Bio 101, Inc.) to remove un-lysed spores.

Automated DNA extraction of specimens was performed utilizing the MagNA Pure LC and Total Nucleic Acid Extraction Kit I. (Roche Life Sciences). Kit I contains wash buffers for removing PCR inhibitors, salts and proteins, lyses and binding solution, proteinase K, magnetic beads for binding of DNA and an elution buffer. Post extraction filtering in a spin module column was also performed as stated above.

BoNT real-time PCR primer and probes

The BoNT real-time PCR assay targets the toxin genes of C. botulinum toxin types A, B, E, F, and C. baratii F. Primers and probes were designed using Primer Express v.3.0 (Life Technologies). All primer and probes were manufactured by Integrated DNA Technologies, Inc. (Table 1). GenBank accession numbers for the original sequences used to generate these primer and probe sets in Primer Express are: X73423-toxin type A; AF295926-toxin type B; AB039264-toxin type E; X81714-toxin type F; and X68262-baratii toxin type F.