**2. Technology and target**

#### **2.1.** *qPCR*

**•** "In all the circumstances and taking into account the standard which might be expected of a doctor practising in the same field of medicine in similar circumstances in or around 1996-1998, the Panel concluded that Dr Wakefield's misconduct not only collectively amounts to serious professional misconduct, over a timeframe from 1996 to 1999, but also, when considered individually, constitutes multiple separate instances of serious profes‐ sional misconduct. Accordingly the Panel finds Dr Wakefield guilty of serious professio‐

**Figure 1.** The polymerase chain reaction, a method for copying the same stretch of DNA several million-fold. **A.** A PCR reaction consists of double stranded DNA, two short DNA molecules ("primers") whose sequence is complementary to opposite strands of the DNA, a DNA synthesis enzyme ("*Taq* polymerase") and four nucleotide building blocks ("dNTPs"). The reaction mixture is heated to 95ºC to dissociate the sample DNA strands, then cooled to around 55ºC to allow the two primers to bind to their targets on the individual strands. Next, *Taq* polymerase makes two new strands of DNA at its optimal temperature of around 70ºC, using the original strands as templates, hence duplicating the original DNA. This procedure is repeated many times, leading to more than one billion exact copies of the original DNA segment. These can be detected by running the samples on a gel and staining with a DNA-binding dye. **B.** qPCR obviates the need for gel electrophoresis by using fluorescence to detect copied DNA. The qPCR method used for the detection of MeV uses a target-specific DNA molecule ("probe") that has a fluorescent dye at one end (R) and a quencher (Q) on the other. In the absence of target, the quencher prevents the dye from emitting light. In the pres‐ ence of target, the probe binds to its target and is degraded by the *Taq* polymerase. This separates the fluorescent label and the quencher and so results in the emission of light. Both the PCR and light detection are automated and

nal misconduct" and

82 Recent Advances in Autism Spectrum Disorders - Volume I

detected in a single step by a dedicated instrument.

A key attraction of qPCR technology is its apparent simplicity: an assay consisting of combining oligonucleotides, PCR enzyme and buffer with a nucleic acid template to pro‐ duce a qPCR reaction is perceived as undemanding. This practical simplicity is comple‐ mented by the absence of any requirement for post-assay handling, as well as the development of user-friendly data analysis software that makes data generation and vis‐ ualisation in the shape of amplification plots remarkably simple. Indeed, qPCR is often described as a mature technology and as the "gold standard" for nucleic acid quantifica‐ tion. Whilst it is true that the technology is capable of exquisite sensitivity and specifici‐ ty, coupled with high reproducibility and accuracy, it is essential to understand that qPCR assays are made up of numerous, often divergent protocols that use different in‐ struments, enzymes, buffers and non-identical targets.

Whilst qPCR is the method of choice for the detection of DNA, the enzyme (*Taq* polymerase) used to copy the DNA template does not work well with RNA. Hence, if the amplification target is RNA, as it is for MeV, an enzymatic RNA-to-DNA conversion (reverse transcrip‐ tion, RT) step must be carried out before the DNA copying step can specifically amplify the target of interest. This variant of the qPCR is termed reverse-transcription (RT)-qPCR and the principle of this reaction is simple: RNA is reverse transcribed into single stranded DNA, either in a separate reaction or as a "one tube" assay that uses a different enzyme (*Tth* polymerase), as was the case with the experiments discussed below. There are several detec‐ tion chemistries, but in this case a very specific and widely used probe-based method was used (Figure 1B). Importantly, the use of fluorescent reporter molecules permits concurrent target amplification, detection and quantification as the assay proceeds [4]. Fluorescence is detected using dedicated qPCR instruments, which have (i) a controllable heating block that can hold a variety of temperatures and rapidly change between them, (ii) an excitation light source to excite the fluorochrome, (iii) a detector to register photon emissions and (iv) soft‐ ware that allows analysis of the data. Fluorescence emissions are collected from each sample tube and the levels of background fluorescence detected by the fluorimeter module of the qPCR instrument are established, providing a baseline that defines the sensitivity of the in‐ strument. Instrument-specific algorithms are used to define a fluorescence threshold for each sample. Finally, the algorithm searches the data from each sample for a point that ex‐ ceeds the baseline and plots a characteristic amplification plot.

The purpose of this chapter is to demonstrate that the qPCR data claiming to detect MeV in

Why There Is no Link Between Measles Virus and Autism

http://dx.doi.org/10.5772/52844

85

**•** Absence of transparency: the key publication shows no data; hence an expert reader can‐

**•** Lack of reproducibility: the data could not be duplicated by several independent investi‐

The only conclusion possible is that the assays were detecting contaminating DNA. Since MeV is an RNA-only virus and never exists in DNA form, these data must be ignored and it it is my opinion that the authors should withdraw this publication from the peer-reviewed

**Figure 2.** MeV life cycle. The virus attaches to the surface of a host cell, the viral envelope fuses to the plasma mem‐ brane and the nucleocapsid is released into the cell. Negative-sense genomic RNA is transcribed into individual mes‐ senger RNAs as well as a full-length positive-sense RNA template, which is used to create negative-sense RNA. Viral

proteins are translated, assembled around the negative sense RNA and new viruses bud from the cells.

the intestine of autistic children are unreliable and meaningless because of

**•** Unreliable techniques and protocols: analysis of the qPCR data was incorrect

**•** Disregard for controls: obvious evidenceof extensive contamination was disregarded

not evaluate the reliability of its conclusions

gators

literature.

Increases in fluorescent signal are proportional to the amount of DNA produced during each PCR cycle and produce a characteristic quantification cycle (Cq) for every test. As a re‐ sult, the more initial target there is, the sooner the instrument can detect the fluorescence and the lower the Cq. Conversely, a higher Cq denotes less initial target. This correlation be‐ tween fluorescence and amount of amplified product permits accurate quantification of tar‐ get molecules over a wide dynamic range in the presence of suitable standards.

The consistency and reliability of RT-qPCR assays depends on the appropriate execution of a number of steps, principally those involving sample selection, template quality, assay de‐ sign and data analysis.

#### **2.2. Measles virus**

MeVs, from the family Paramyxoviridae, genus Morbillivirus, have a single negativestrand RNA genome enclosed in a viral envelope associated with three proteins: the ma‐ trix (M) lining the inner surface of the envelope and the fusion (F) and haemagglutinin (H) transmembrane proteins. Negative sense means that the RNA is complementary to mRNA and must be copied into the complementary plus-sense mRNA before proteins can be made. Thus, besides needing to code for an RNA-dependent RNA-polymerase, these viruses also need to package it in the virion so that they can make mRNAs upon in‐ fecting the cell. The MeV RNA-dependent RNA polymerase generates a full-length posi‐ tive copy of their genome, the "replicative intermediate" as well as individual RNA copies that serve as mRNA for individual virus-specific proteins (Figure 2). Importantly, at no stage of its replication cycle is MeV RNA ever reverse transcribed into DNA, *i.e.* MeV does not exist as a DNA molecule, a fact that was established in 1964. This is a criti‐ cal issue, since if it can be demonstrated that a test is amplifying DNA and not RNA, this provides indisputable proof of contamination.

#### **2.3. What makes a publication credible?**

Before describing the experiments in detail, it is worth reiterating what is expected of a plau‐ sible scientific publication. Its credibility depends on a number of conditions that include


This is especially so for publications that utilises qPCR, since its sensitivity makes experi‐ ments susceptible to contamination, which leads to the reporting of false positive results. The purpose of this chapter is to demonstrate that the qPCR data claiming to detect MeV in the intestine of autistic children are unreliable and meaningless because of


strument. Instrument-specific algorithms are used to define a fluorescence threshold for each sample. Finally, the algorithm searches the data from each sample for a point that ex‐

Increases in fluorescent signal are proportional to the amount of DNA produced during each PCR cycle and produce a characteristic quantification cycle (Cq) for every test. As a re‐ sult, the more initial target there is, the sooner the instrument can detect the fluorescence and the lower the Cq. Conversely, a higher Cq denotes less initial target. This correlation be‐ tween fluorescence and amount of amplified product permits accurate quantification of tar‐

The consistency and reliability of RT-qPCR assays depends on the appropriate execution of a number of steps, principally those involving sample selection, template quality, assay de‐

MeVs, from the family Paramyxoviridae, genus Morbillivirus, have a single negativestrand RNA genome enclosed in a viral envelope associated with three proteins: the ma‐ trix (M) lining the inner surface of the envelope and the fusion (F) and haemagglutinin (H) transmembrane proteins. Negative sense means that the RNA is complementary to mRNA and must be copied into the complementary plus-sense mRNA before proteins can be made. Thus, besides needing to code for an RNA-dependent RNA-polymerase, these viruses also need to package it in the virion so that they can make mRNAs upon in‐ fecting the cell. The MeV RNA-dependent RNA polymerase generates a full-length posi‐ tive copy of their genome, the "replicative intermediate" as well as individual RNA copies that serve as mRNA for individual virus-specific proteins (Figure 2). Importantly, at no stage of its replication cycle is MeV RNA ever reverse transcribed into DNA, *i.e.* MeV does not exist as a DNA molecule, a fact that was established in 1964. This is a criti‐ cal issue, since if it can be demonstrated that a test is amplifying DNA and not RNA, this

Before describing the experiments in detail, it is worth reiterating what is expected of a plau‐ sible scientific publication. Its credibility depends on a number of conditions that include

This is especially so for publications that utilises qPCR, since its sensitivity makes experi‐ ments susceptible to contamination, which leads to the reporting of false positive results.

get molecules over a wide dynamic range in the presence of suitable standards.

ceeds the baseline and plots a characteristic amplification plot.

84 Recent Advances in Autism Spectrum Disorders - Volume I

sign and data analysis.

provides indisputable proof of contamination.

**2.3. What makes a publication credible?**

**•** Reliability of protocols and techniques

**•** Inclusion of appropriate controls

**•** Transparency of reporting

**•** Reproducibility of the data

**2.2. Measles virus**


The only conclusion possible is that the assays were detecting contaminating DNA. Since MeV is an RNA-only virus and never exists in DNA form, these data must be ignored and it it is my opinion that the authors should withdraw this publication from the peer-reviewed literature.

**Figure 2.** MeV life cycle. The virus attaches to the surface of a host cell, the viral envelope fuses to the plasma mem‐ brane and the nucleocapsid is released into the cell. Negative-sense genomic RNA is transcribed into individual mes‐ senger RNAs as well as a full-length positive-sense RNA template, which is used to create negative-sense RNA. Viral proteins are translated, assembled around the negative sense RNA and new viruses bud from the cells.
